Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma
Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma
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DOI：
10.1016/s0021-9258(19)34116-x
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发表时间：
1990-03
期刊：
影响因子：
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通讯作者：
T. Chijiwa;A. Mishima;M. Hagiwara;M. Sano;Kyozo Hayashi;Tsutomu Inoue;Naito Kenji;T. Toshioka
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文献类型：
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作者：
T. Chijiwa;A. Mishima;M. Hagiwara;M. Sano;Kyozo Hayashi;Tsutomu Inoue;Naito Kenji;T. Toshioka
新合成的异喹啉磺酰胺H-89(N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide)，对环磷酸腺苷依赖的蛋白激酶(蛋白激酶A)有很强的选择性抑制作用，抑制常数为0.048+/-0.008微米。H-89对cGMP依赖的蛋白激酶(蛋白激酶G)、钙/磷脂依赖的蛋白激酶(C)、酪蛋白激酶I和酪蛋白激酶II、肌球蛋白轻链激酶和钙/钙调蛋白依赖的蛋白激酶II的Ki分别为136.7+/-17.0、28.3+/-17.5和29.7+/-8.1微米。动力学分析表明，H-89以与ATP竞争的方式抑制蛋白激酶A。为了研究蛋白激酶A在PC12细胞突起生长中的作用，H-89与神经生长因子(NGF)、Forsklin或二丁酰cAMP一起应用。H-可剂量依赖性地抑制Forsklin诱导的PC12D细胞内cAMP水平，但对NGF诱导的蛋白磷酸化无明显抑制作用。H-89还能显著抑制Forskolin诱导的PC12D细胞突起生长。当H-89在加入二丁酰cAMP之前加入时，这种抑制作用也发生了。H-89(30微米)可显著抑制PC12D细胞裂解产物中cAMP依赖的组蛋白IIb的磷酸化活性，但不影响其他蛋白的磷酸化活性，如cGMP依赖的组蛋白IIb的磷酸化活性、钙/磷脂依赖的组蛋白IIis的磷酸化活性、钙/钙调蛋白依赖的肌球蛋白轻链的磷酸化活性和α-酪蛋白的磷酸化活性。然而，该蛋白激酶A抑制剂不能抑制NGF诱导的PC12D细胞突起生长。因此，Forskolin和Dbuyryl cAMP诱导的轴突生长显然是由蛋白激酶A介导的，而NGF诱导的轴突生长则是通过不依赖于蛋白激酶A的途径介导的。
A newly synthesized isoquinolinesulfonamide, H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide), was shown to have a potent and selective inhibitory action against cyclic AMP-dependent protein kinase (protein kinase A), with an inhibition constant of 0.048 +/- 0.008 microM. H-89 exhibited weak inhibitory action against other kinases and Ki values of the compound for these kinases, including cGMP-dependent protein kinase (protein kinase G), Ca2+/phospholipid-dependent protein kinase (protein kinase C), casein kinase I and II, myosin light chain kinase, and Ca2+/calmodulin-dependent protein kinase II were 0.48 +/- 0.13, 31.7 +/- 15.9, 38.3 +/- 6.0, 136.7 +/- 17.0, 28.3 +/- 17.5, and 29.7 +/- 8.1 microM, respectively. Kinetic analysis indicated that H-89 inhibits protein kinase A, in competitive fashion against ATP. To examine the role of protein kinase A in neurite outgrowth of PC12 cells, H-89 was applied along with nerve growth factor (NGF), forskolin, or dibutyryl cAMP. Pretreatment with H-89 led to a dose-dependent inhibition of the forskolin-induced protein phosphorylation, with no decrease in intracellular cyclic AMP levels in PC12D cells, and the NGF-induced protein phosphorylation was not not inhibited. H-89 also significantly inhibited the forskolin-induced neurite outgrowth from PC12D cells. This inhibition also occurred when H-89 was added before the addition of dibutyryl cAMP. Pretreatment of PC12D cells with H-89 (30 microM) inhibited significantly cAMP-dependent histone IIb phosphorylation activity in cell lysates but did not affect other protein phosphorylation activity such as cGMP-dependent histone IIb phosphorylation activity, Ca2+/phospholipid-dependent histone IIIs phosphorylation activity, Ca2+/calmodulin-dependent myosin light chain phosphorylation activity, and alpha-casein phosphorylation activity. However, this protein kinase A inhibitor did not inhibit the NGF-induced neurite outgrowth from PC12D cells. Thus, the forskolin- and dibutyryl cAMP-induced neurite outgrowth is apparently mediated by protein kinase A while the NGF-induced neurite outgrowth is mediated by a protein kinase A-independent pathway.