Niwa M, Hara A, Kanamori Y, Matsuno H, Kozawa O, Yoshimi N, Mori H, Uematsu T
Department of Pharmacology, Gifu University School of Medicine, Japan.
Eur J Pharmacol. 1999 Apr 23;371(1):59-67. doi: 10.1016/s0014-2999(99)00145-4.
Treatment of neutrophils with tumor necrosis factor-alpha (TNF-alpha) in the presence of cycloheximide induced apoptosis within 3 h, as evaluated by the occurrence of morphological nuclear changes characteristic of apoptosis. Pretreatment of neutrophils with dibutyryl cyclic AMP (dbcAMP) suppressed the TNF-alpha/cycloheximide-induced apoptosis in neutrophils in a concentration-dependent manner, while dbcAMP by itself did not induce any morphological changes. Forskolin, or a phosphodiesterase inhibitor, also produced a concentration-dependent inhibition on apoptosis. This inhibition by dbcAMP was completely reversed by pretreatment with the protein kinase A inhibitor, N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinoline sulphonamide (H-89). DbcAMP also inhibited the TNF-alpha/cycloheximide-induced activation of caspase-3, but it had no effect on the activation of caspase-8 in human neutrophils. Furthermore, dbcAMP did not directly inhibit activated caspase-3 activity. Inhibitor of protein kinase C, phosphatidylcholine-specific phospholipase C, tyrosine kinase, nitric oxide synthase, or granulocyte colony-stimulating factor or granulocyte monocyte colony-stimulating factor did not affect apoptosis. These results indicate that the elevation of levels of endogenous intracellular cyclic AMP and subsequent activation of protein kinase A play a crucial role in the prevention of apoptosis triggered by TNF-alpha/cycloheximide in human neutrophils, and that the possible target of cyclic AMP is a product in the metabolic pathway between caspase-8 and caspase-3.
在放线菌酮存在的情况下，用肿瘤坏死因子-α（TNF-α）处理中性粒细胞会在3小时内诱导细胞凋亡，这可通过凋亡特有的形态学核变化来评估。用二丁酰环磷腺苷（dbcAMP）预处理中性粒细胞可浓度依赖性地抑制TNF-α/放线菌酮诱导的中性粒细胞凋亡，而dbcAMP本身不会诱导任何形态学变化。福斯高林或磷酸二酯酶抑制剂也对细胞凋亡产生浓度依赖性抑制作用。蛋白激酶A抑制剂N-[2-(对溴肉桂酰胺基)乙基]-5-异喹啉磺酰胺（H-89）预处理可完全逆转dbcAMP的这种抑制作用。dbcAMP还抑制TNF-α/放线菌酮诱导的半胱天冬酶-3激活，但对人中性粒细胞中半胱天冬酶-8的激活没有影响。此外，dbcAMP不会直接抑制激活的半胱天冬酶-3活性。蛋白激酶C、磷脂酰胆碱特异性磷脂酶C、酪氨酸激酶、一氧化氮合酶、粒细胞集落刺激因子或粒细胞-巨噬细胞集落刺激因子的抑制剂均不影响细胞凋亡。这些结果表明，内源性细胞内环磷腺苷水平的升高以及随后蛋白激酶A的激活在预防TNF-α/放线菌酮触发的人中性粒细胞凋亡中起关键作用，并且环磷腺苷的可能作用靶点是半胱天冬酶-8和半胱天冬酶-3之间代谢途径中的一种产物。
