Rodríguez-Pascual F, Ferrero R, Miras-Portugal M T, Torres M
Biochemistry Department, Veterinary Faculty, Madrid, Spain.
Arch Biochem Biophys. 1999 Jun 15;366(2):207-14. doi: 10.1006/abbi.1999.1199.
The phosphorylation of the enzyme tyrosine hydroxylase by the cGMP pathway was investigated in chromaffin cells from the bovine adrenal medulla. The nitric oxide donor, sodium nitroprusside, and the natriuretic peptide, C-type natriuretic peptide, which are able to increase cGMP levels and cGMP-dependent protein kinase activity, produced significant increases in the phosphorylation level of tyrosine hydroxylase in a time- and concentration-dependent manner. The pretreatment of the cells with the soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one blocked the effect of sodium nitroprusside. This result indicates that cGMP production by this enzyme mediated this effect. Experiments performed with a cGMP-dependent protein kinase inhibitor, the Rp-isomer of 8-(4-chlorophenylthio)-cyclic guanosine monophosphorothioate, which blocked the effects of both sodium nitroprusside and C-type natriuretic peptide, demonstrated that the phosphorylation increases evoked by both compounds were mediated by the activation of cGMP-dependent protein kinase. In cells incubated with the adenylyl cyclase activator, forskolin, an increase in the phosphorylation level of the tyrosine hydroxylase was also found. When cells were treated simultaneously with forskolin and sodium nitroprusside or C-type natriuretic peptide, an additive effect on tyrosine hydroxylase phosphorylation was not observed. This suggests that cAMP- and cGMP-dependent protein kinases may phosphorylate the same amino acid residues in the enzyme. Western blot analysis of soluble extracts from chromaffin cells detected specific immunoreactivity for two different commercial antibodies raised against cGMP-dependent protein kinase (both Ialpha and Ibeta isoforms). Electrophoretic mobility correlates with that of purified PKG Ialpha. Because the phosphorylation of the tyrosine hydroxylase correlates with increases in its enzymatic activity and thus with augmentation in the cell capacity to synthesize catecholamines, our results indicate that a cGMP-based second messenger pathway participates in catecholamine biosynthesis regulation in chromaffin cells, a mechanism which may be widespread in other catecholamine-synthesizing cells.
在来自牛肾上腺髓质的嗜铬细胞中研究了cGMP途径对酪氨酸羟化酶的磷酸化作用。一氧化氮供体硝普钠和利钠肽C型利钠肽能够提高cGMP水平和cGMP依赖性蛋白激酶活性，它们能以时间和浓度依赖性方式使酪氨酸羟化酶的磷酸化水平显著升高。用可溶性鸟苷酸环化酶抑制剂1H-[1,2,4]恶二唑并[4,3-a]喹喔啉-1-酮预处理细胞可阻断硝普钠的作用。该结果表明该酶产生的cGMP介导了这一效应。用cGMP依赖性蛋白激酶抑制剂8-(4-氯苯硫基)-环鸟苷单磷酸硫代酯的Rp异构体进行的实验阻断了硝普钠和C型利钠肽的作用，这表明这两种化合物引起的磷酸化增加是由cGMP依赖性蛋白激酶的激活介导的。在用腺苷酸环化酶激活剂福斯可林孵育的细胞中，也发现酪氨酸羟化酶的磷酸化水平升高。当细胞同时用福斯可林和硝普钠或C型利钠肽处理时，未观察到对酪氨酸羟化酶磷酸化的累加效应。这表明cAMP依赖性和cGMP依赖性蛋白激酶可能使该酶中的相同氨基酸残基磷酸化。对嗜铬细胞可溶性提取物的蛋白质免疫印迹分析检测到针对cGMP依赖性蛋白激酶（Iα和Iβ亚型）的两种不同商业抗体的特异性免疫反应性。电泳迁移率与纯化的PKG Iα的迁移率相关。由于酪氨酸羟化酶的磷酸化与其酶活性的增加相关，进而与细胞合成儿茶酚胺的能力增强相关，我们的结果表明基于cGMP的第二信使途径参与嗜铬细胞中儿茶酚胺生物合成的调节，这一机制可能在其他儿茶酚胺合成细胞中广泛存在。
