Barile F A, Guzowski D E, Ripley C, Siddiqi Z A, Bienkowski R S
Department of Natural Sciences, York College/City University of New York, Jamaica 11451.
Arch Biochem Biophys. 1990 Jan;276(1):125-31. doi: 10.1016/0003-9861(90)90018-t.
The objective of this work was to characterize basal degradation of newly synthesized collagen in human fetal lung fibroblasts. Analysis of 22 separate determinations showed that in cells incubated under normal conditions, the level of intracellular degradation was normally distributed with a mean of 15.2% and a standard deviation of 2.6%. Within each experiment, however, the uncertainty (standard deviation) in determining degradation was very small, usually less than 1.5%. Consideration of the large variation between experiments and the ability of our analytic technique to detect small, but "statistically significant," differences between groups within the same experiment led us to formulate two criteria for determining whether degradation measured in cultures exposed to some agent differs in a "biologically significant" way from degradation measured in control cultures. These criteria were used to evaluate the effects of the following proteinase inhibitors on basal degradation: NH4Cl, which increases the pH of subcellular compartments that are normally acidic; and leupeptin and Na-p-tosyl-L-lysine chloromethyl ketone (TLCK), which are inhibitors of lysosomal cathepsins (B and L) that degrade collagen. NH4Cl (16 mM) lowered degradation to an extent that was both statistically and biologically significant, but neither leupeptin nor TLCK affected degradation. The effect of NH4Cl on degradation was independent of its inhibitory effects on production of collagen, protein, and ATP. These results suggest that basal degradation occurs in, or beyond, an acidic (i.e., NH4Cl-sensitive) but nonlysosomal compartment of the cell, and that NH4Cl inhibits processing within, or transport to, that compartment. This is the first report of an agent that inhibits basal degradation of newly synthesized collagen in soft tissue fibroblasts.
这项工作的目的是表征人胎儿肺成纤维细胞中新合成胶原蛋白的基础降解情况。对22次独立测定的分析表明，在正常条件下培养的细胞中，细胞内降解水平呈正态分布，平均值为15.2%，标准差为2.6%。然而，在每个实验中，测定降解时的不确定性（标准差）非常小，通常小于1.5%。考虑到不同实验之间的巨大差异以及我们的分析技术检测同一实验中不同组之间微小但“具有统计学意义”差异的能力，我们制定了两个标准来确定在暴露于某种试剂的培养物中测得的降解与在对照培养物中测得的降解是否在“生物学上有显著差异”。这些标准用于评估以下蛋白酶抑制剂对基础降解的影响：NH4Cl，它会提高通常呈酸性的亚细胞区室的pH值；以及亮抑酶肽和对甲苯磺酰-L-赖氨酸氯甲基酮（TLCK），它们是降解胶原蛋白的溶酶体组织蛋白酶（B和L）的抑制剂。NH4Cl（16 mM）将降解降低到在统计学和生物学上均有显著意义的程度，但亮抑酶肽和TLCK均未影响降解。NH4Cl对降解的影响与其对胶原蛋白、蛋白质和ATP产生的抑制作用无关。这些结果表明，基础降解发生在细胞的酸性（即对NH4Cl敏感）但非溶酶体区室之内或之外，并且NH4Cl抑制该区域内的加工或向该区域的转运。这是关于一种抑制软组织成纤维细胞中新合成胶原蛋白基础降解的试剂的首次报道。
