Stimulation and priming of protein kinase C translocation by a Ca2+ transient-independent mechanism. Studies in human neutrophils challenged with platelet-activating factor and other receptor agonists.
N-Formyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 stimulate human polymorphonuclear neutrophils (PMN) to translocate protein kinase C from the cytosol to plasmalemma as judged by their abilities to increase PMN binding of and receptor numbers for [3H]phorbol dibutyrate [( 3H]PDB) (O'Flaherty, J.T., Jacobson, D.P., Redman, J.F., and Rossi, A.G. (1990) J. Biol. Chem. 265, 9146-9152). Platelet-activating factor (PAF) had these same effects. Moreover, two potent PAF analogs (but not an inactive analog) increased [3H]PDB binding; a PAF antagonist blocked responses to PAF without altering those to fMLP; and PMN treated with PAF became desensitized to PAF while retaining sensitivity to fMLP. Indeed, PMN incubated with 1-100 nM PAF for 5-40 min had markedly enhanced [3H]PDB binding responses to fMLP. PAF thus acted through its receptors to stimulate and prime protein kinase C translocation. Its effects, however, did not necessarily proceed by a standard mechanism: Ca2(+)-depleted PMN failed to raise Fura-2-monitored cytosolic Ca2+ concentrations [( Ca2+]i), yet increased [3H]PDB binding and receptor numbers almost normally after PAF challenge. PAF also primed Ca2(+)-depleted PMN to fMLP. Nevertheless, [3H]PDB binding responses to PAF were blocked in PMN loaded with Ca2+ chelators, viz. Quin 2, Fura-2, or 5,5'-dimethyl-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Exogenous Ca2+ reversed Quin 2 inhibition, and a weak chelator 4,4'-difluoro-BAPTA, lacked inhibitory actions. The chelators similarly influenced fMLP and leukotriene B4. Thus, PMN can by-pass [Ca2+]i to translocate protein kinase C. They may achieve this using a regulatable pool of Ca2+ that evades conventional [Ca2+]i monitors or a signal that needs cell Ca2+ to form and/or act. This signal may mediate function in Ca2(+)-depleted cells, the actions of [Ca2+]i-independent stimuli, cell priming, and protein kinase C movements that otherwise seem [Ca2+]i-induced.
O'Flaherty
J T
JT
Department of Medicine, Wake Forest University Medical Center, Winston-Salem, North Carolina 27103.
Redman
J F
JF
Jacobson
D P
DP
Rossi
A G
AG
D002118:Calcium / Q000502:physiology*; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism; D009504:Neutrophils / Q000201:enzymology*; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D010972:Platelet Activating Factor / Q000494:pharmacology*; D010980:Platelet Membrane Glycoproteins; D011493:Protein Kinase C / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000502:physiology; D043562:Receptors, G-Protein-Coupled
D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C039351:platelet activating factor receptor; D015240:Phorbol 12,13-Dibutyrate; D009240:N-Formylmethionine Leucyl-Phenylalanine; D011493:Protein Kinase C; D002118:Calcium