O'Flaherty J T, Redman J F, Jacobson D P, Rossi A G
Department of Medicine, Wake Forest University Medical Center, Winston-Salem, North Carolina 27103.
J Biol Chem. 1990 Dec 15;265(35):21619-23.
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.
N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸（fMLP）和白三烯B4可刺激人多形核中性粒细胞（PMN），使蛋白激酶C从胞质溶胶转位至质膜，这可通过它们增加PMN对[3H]佛波醇二丁酸酯[（3H）PDB]的结合及受体数量的能力来判断（奥弗莱厄蒂，J.T.，雅各布森，D.P.，雷德曼，J.F.，和罗西，A.G.（1990年）《生物化学杂志》265卷，9146 - 9152页）。血小板活化因子（PAF）也有同样的作用。此外，两种强效PAF类似物（但无活性类似物）可增加[3H]PDB结合；一种PAF拮抗剂可阻断对PAF的反应，而不改变对fMLP的反应；用PAF处理的PMN对PAF脱敏，同时保留对fMLP的敏感性。实际上，用1 - 100 nM PAF孵育5 - 40分钟的PMN对fMLP的[3H]PDB结合反应明显增强。因此，PAF通过其受体发挥作用，刺激并引发蛋白激酶C转位。然而，其作用不一定通过标准机制进行：用钙离子载体A23187处理使细胞内钙离子（Ca2 +）耗竭的PMN未能提高用Fura - 2监测的胞质Ca2 +浓度[（Ca2 +）i]，但在PAF刺激后，[3H]PDB结合及受体数量几乎正常增加。PAF还使Ca2 +耗竭细胞的PMN对fMLP致敏。然而，在加载有Ca2 +螯合剂（即喹啉2、Fura - 2或5,5'-二甲基-1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸（BAPTA））的PMN中，对PAF的[3H]PDB结合反应被阻断。外源性Ca2 +可逆转喹啉2的抑制作用，而一种弱螯合剂4,4'-二氟BAPTA则无抑制作用。这些螯合剂对fMLP和白三烯B4也有类似影响。因此，PMN可绕过（Ca2 +）i使蛋白激酶C转位。它们可能通过利用一个可调节的Ca2 +池来实现这一点，该Ca2 +池可避开传统的（Ca2 +）i监测器，或者通过一个需要细胞Ca2 +来形成和/或起作用的信号。该信号可能介导Ca2 +耗竭细胞中的功能、不依赖（Ca2 +）i的刺激的作用、细胞致敏以及蛋白激酶C的移动，否则这些似乎是由（Ca2 +）i诱导的。
