Sha'afi R I, Molski T F, Gomez-Cambronero J, Huang C K
Department of Physiology, University of Connecticut Health Center, Farmington 06032.
J Leukoc Biol. 1988 Jan;43(1):18-27. doi: 10.1002/jlb.43.1.18.
The aim for the present studies is to examine the relationship between the phosphorylation of the 47-kDa protein and some neutrophil responses such as degranulation, the synergistic effect of PMA on calcium ionophore-induced degranulation, superoxide generation, and the priming of the oxidative burst produced by the chemotactic factor fMet-Leu-Phe and phorbol 12-myristate 13-acetate (PMA). Incubation of neutrophils with the protein kinase inhibitor 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H-7) inhibits the phosphorylation of the 47-kDa protein produced by PMA and fMet-Leu-Phe but does not affect lysozyme release induced by the same stimuli or fMet-Leu-Phe-induced N-acetyl-beta-glucosaminidase release. Furthermore, the synergistic effect of PMA on the A23187-induced degranulation is not inhibited by H-7. Also, pretreatment of the cells with H-7 inhibits superoxide production produced by PMA but not by fMet-Leu-Phe. The inhibitory effect of H-7 is more pronounced on the rate than on the extent of PMA-induced superoxide release. On the other hand, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride, HA-1004, a less potent protein kinase inhibitor, has no inhibitory effect on superoxide generation produced by fMet-Leu-Phe or PMA. In the case of superoxide production, the addition of low concentrations of PMA to rabbit neutrophils primes these cells to the subsequent stimulation by fMet-Leu-Phe, dramatically increasing the effect. Conversely, low concentrations of fMet-Leu-Phe prime the cells to PMA stimulation. The stimulation by PMA of cells primed with fMet-Leu-Phe is inhibited by H-7. Moreover, the priming effect by PMA is also inhibited by H-7. This inhibition is less pronounced at a low concentration of PMA. On the other hand, in the case of human neutrophils, the priming effect of PMA is not inhibited by H-7. These results suggest several points. First, phosphorylation of the protein identified on two-dimensional gel electrophoresis as having a molecular weight of 47 kDa and PI of 4.9 is not necessary for degranulation produced by either PMA or fMet-Leu-Phe. Second, the phosphorylation of the 47-kDa protein is not necessary for superoxide generation, at least in the case of fMet-Leu-Phe and possibly for other stimuli. Third, in rabbit neutrophils, both the priming and stimulation of superoxide production with PMA are inhibited by H-7. In human neutrophils, the priming by PMA is not affected by H-7.(ABSTRACT TRUNCATED AT 250 WORDS)
本研究的目的是检测47 kDa蛋白的磷酸化与一些中性粒细胞反应之间的关系，这些反应包括脱颗粒、佛波酯（PMA）对钙离子载体诱导的脱颗粒的协同作用、超氧化物生成，以及趋化因子fMet-Leu-Phe和佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）引发的氧化爆发。用蛋白激酶抑制剂1-（5-异喹啉磺酰基）-2-甲基哌嗪（H-7）孵育中性粒细胞，可抑制PMA和fMet-Leu-Phe诱导的47 kDa蛋白的磷酸化，但不影响相同刺激诱导的溶菌酶释放或fMet-Leu-Phe诱导的N-乙酰-β-葡萄糖苷酶释放。此外，H-7不抑制PMA对A23187诱导的脱颗粒的协同作用。同样，用H-7预处理细胞可抑制PMA产生的超氧化物生成，但不抑制fMet-Leu-Phe产生的超氧化物生成。H-7对PMA诱导的超氧化物释放速率的抑制作用比对其释放程度的抑制作用更明显。另一方面，较弱的蛋白激酶抑制剂盐酸N-（2-胍基乙基）-5-异喹啉磺酰胺（HA-1004）对fMet-Leu-Phe或PMA产生的超氧化物生成没有抑制作用。在超氧化物生成方面，向兔中性粒细胞中添加低浓度的PMA可使这些细胞对随后的fMet-Leu-Phe刺激产生预刺激作用，显著增强效应。相反，低浓度的fMet-Leu-Phe可使细胞对PMA刺激产生预刺激作用。用fMet-Leu-Phe预刺激的细胞对PMA的刺激作用被H-7抑制。此外，PMA的预刺激作用也被H-7抑制。在低浓度PMA时，这种抑制作用不太明显。另一方面，在人中性粒细胞中，PMA的预刺激作用不受H-7抑制。这些结果表明了几点。第一，二维凝胶电泳鉴定出的分子量为47 kDa、等电点为4.9的蛋白磷酸化对于PMA或fMet-Leu-Phe诱导的脱颗粒不是必需的。第二，47 kDa蛋白的磷酸化对于超氧化物生成不是必需的，至少在fMet-Leu-Phe的情况下可能对其他刺激也不是必需的。第三，在兔中性粒细胞中，PMA引发和刺激超氧化物生成均被H-7抑制。在人中性粒细胞中，PMA的引发作用不受H-7影响。（摘要截短至250字）
