Azuma E K, Kitagawa S, Yuo A, Mizoguchi H, Umezawa K, Takaku F, Saito M
Division of Hemopoiesis, Jichi Medical School, Tochigi, Japan.
Biochim Biophys Acta. 1993 Nov 7;1179(2):213-23. doi: 10.1016/0167-4889(93)90144-e.
Human neutrophils maximally stimulated with the optimal concentration (100 ng/ml) of phorbol myristate acetate (PMA), a direct activator of protein kinase C (PKC), for 5 min at 37 degrees C did not respond with superoxide (O2-) release to the later addition of PMA itself or the Ca2+ ionophore ionomycin. However, these cells did respond with enhanced release of O2- to the later addition of N-formyl-methionyl-leucyl-phenylalanine (FMLP) or concanavalin A (Con A). In these PMA-pretreated cells, an increase in cytoplasmic free Ca2+ ([Ca2+]i) induced by ionomycin was unaffected, whereas that induced by FMLP was inhibited by 50-60% and that induced by Con A was completely abolished. A 42-kDa protein was predominantly and consistently tyrosine-phosphorylated by FMLP, PMA and ionomycin with the different kinetics according to the stimuli. The dose-response curves showed that tyrosine phosphorylation and O2- release were stimulated in parallel by PMA, whereas tyrosine phosphorylation and an increase in [Ca2+]i, but not O2- release, were stimulated in parallel by FMLP or ionomycin. The potency of inducing tyrosine phosphorylation was ionomycin > FMLP = PMA, whereas the potency of triggering of O2- release was PMA > ionomycin = FMLP. UCN-01, a PKC inhibitor, inhibited O2- release and tyrosine phosphorylation induced by PMA, but not by FMLP or ionomycin. In contrast, pertussis toxin inhibited O2- release and tyrosine phosphorylation induced by FMLP, but not by PMA. Tyrosine kinase inhibitors (erbstatin and genistein) inhibited O2- release induced by FMLP, but not by PMA. However, both tyrosine kinase inhibitors did not impair FMLP- or PMA-induced tyrosine phosphorylation of a 42-kDa protein. Increased tyrosine phosphorylation of a 42-kDa protein was also detected in immature myeloid cells (HL-60 cells) stimulated by PMA, but not by ionomycin. These findings suggest that FMLP and Con A trigger the respiratory burst in human neutrophils by activating the definite pathway which include other signals than activation of PKC and an increase in [Ca2+]i; tyrosine phosphorylation of a 42-kDa protein is induced by the PKC-dependent and independent mechanisms according to the stimuli, and the PKC-independent and ionomycin-sensitive mechanism is inoperative in HL-60 cells; and tyrosine phosphorylation of a 42-kDa protein is unlikely to be causally related to activation of the respiratory burst.
用人嗜中性粒细胞，在37℃下用佛波醇肉豆蔻酸酯乙酸酯（PMA，蛋白激酶C（PKC）的直接激活剂）的最佳浓度（100 ng/ml）最大程度刺激5分钟，随后再添加PMA本身或Ca2+离子载体离子霉素时，细胞不会以释放超氧化物（O2-）做出反应。然而，这些细胞确实会对随后添加的N-甲酰甲硫氨酰亮氨酰苯丙氨酸（FMLP）或伴刀豆球蛋白A（Con A）做出反应，O2-释放增强。在这些经PMA预处理的细胞中，离子霉素诱导的细胞质游离Ca2+（[Ca2+]i）增加不受影响，而FMLP诱导的增加被抑制50-60%，Con A诱导的增加则完全消除。一种42 kDa的蛋白质主要且持续地被FMLP、PMA和离子霉素酪氨酸磷酸化，根据刺激的不同，其动力学也不同。剂量反应曲线表明，PMA能同时刺激酪氨酸磷酸化和O2-释放，而FMLP或离子霉素能同时刺激酪氨酸磷酸化和[Ca2+]i增加，但不刺激O2-释放。诱导酪氨酸磷酸化的效力为离子霉素>FMLP = PMA，而触发O2-释放的效力为PMA>离子霉素 = FMLP。PKC抑制剂UCN-01抑制PMA诱导的O2-释放和酪氨酸磷酸化，但不抑制FMLP或离子霉素诱导的。相反，百日咳毒素抑制FMLP诱导的O2-释放和酪氨酸磷酸化，但不抑制PMA诱导的。酪氨酸激酶抑制剂（埃博霉素和染料木黄酮）抑制FMLP诱导的O2-释放，但不抑制PMA诱导的。然而，两种酪氨酸激酶抑制剂都不损害FMLP或PMA诱导的42 kDa蛋白质的酪氨酸磷酸化。在经PMA刺激而非离子霉素刺激的未成熟髓样细胞（HL-60细胞）中也检测到42 kDa蛋白质酪氨酸磷酸化增加。这些发现表明，FMLP和Con A通过激活特定途径触发人嗜中性粒细胞的呼吸爆发，该途径包括除PKC激活和[Ca2+]i增加之外的其他信号；根据刺激的不同，42 kDa蛋白质的酪氨酸磷酸化由PKC依赖性和非依赖性机制诱导，且PKC非依赖性和离子霉素敏感机制在HL-60细胞中不起作用；42 kDa蛋白质的酪氨酸磷酸化不太可能与呼吸爆发的激活存在因果关系。
