Penberthy W T, Dahmer M K
Department of Biochemistry, College of Medicine, University of Tennessee, Memphis 38163.
J Neurochem. 1994 May;62(5):1707-15. doi: 10.1046/j.1471-4159.1994.62051707.x.
Previous studies have demonstrated that bovine chromaffin cells cultured in medium with 10 nM insulin-like growth factor-I (IGF-I) secrete about twofold more catecholamine when exposed to secretory stimuli than do cells cultured without IGF-I. The purpose of this study was to determine whether protein kinase C (PKC) is involved in the effect of IGF-I on secretion from these cells. PKC was down-regulated in the cells by 16-18 h of treatment with beta-phorbol didecanoate (beta-PDD; 100 nM). Such treatment had no effect on high-K(+)-stimulated secretion from cells cultured without IGF-I; however, secretion from cells cultured with IGF-I was reduced to a level comparable to that in cells cultured without the peptide. The inactive isomer, alpha-PDD (100 nM), had no effect on secretion from untreated or IGF-I-treated chromaffin cells. The effect of beta-PDD was time and concentration dependent, with 100 nM beta-PDD producing a maximal effect in 8-10 h. In situ PKC activity measured in permeabilized cells treated with PMA (300 nM) was decreased by approximately 40% by 10 h and was reduced to almost basal levels by 18 h. Immunoblotting experiments demonstrated that both alpha- and epsilon-PKC were lost from the cells with time courses similar to that seen in the in situ PKC assay. Overnight treatment with the PKC inhibitor H7 (100 microM) prevented the enhanced secretion normally seen in IGF-I-treated cells, whereas HA1004 had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)
先前的研究表明，在含有10 nM胰岛素样生长因子-I（IGF-I）的培养基中培养的牛嗜铬细胞，在受到分泌刺激时分泌的儿茶酚胺比在无IGF-I培养基中培养的细胞多约两倍。本研究的目的是确定蛋白激酶C（PKC）是否参与IGF-I对这些细胞分泌的影响。用β-佛波醇二癸酸酯（β-PDD；100 nM）处理细胞16 - 18小时可下调PKC。这种处理对在无IGF-I培养基中培养的细胞的高钾刺激分泌没有影响；然而，在含IGF-I培养基中培养的细胞的分泌减少到与无该肽培养基中培养的细胞相当的水平。无活性异构体α-PDD（100 nM）对未处理或经IGF-I处理的嗜铬细胞的分泌没有影响。β-PDD的作用具有时间和浓度依赖性，100 nM β-PDD在8 - 10小时产生最大效应。在用佛波酯（PMA，300 nM）处理的通透细胞中测得的原位PKC活性在10小时时降低约40%，在18小时时降至几乎基础水平。免疫印迹实验表明，α-和ε-PKC均随时间从细胞中丢失，其时间进程与原位PKC测定中所见相似。用PKC抑制剂H7（100 μM）过夜处理可阻止IGF-I处理的细胞中通常出现的分泌增强，而HA1004没有影响。（摘要截短于250字）
