Inhibition of cyclic GMP-dependent protein kinase-mediated effects by (Rp)-8-bromo-PET-cyclic GMPS
Inhibition of cyclic GMP-dependent protein kinase-mediated effects by (Rp)-8-bromo-PET-cyclic GMPS
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DOI：
10.1111/j.1476-5381.1995.tb15112.x
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发表时间：
1995-12-01
影响因子：
7.3
通讯作者：
Bucher, B
中科院分区：
文献类型：
--
作者：
Butt, E;Pohler, D;Bucher, B
1利用纯化的蛋白激酶研究了8-溴- β苯基-1、n -2-乙基鸟苷3‘、5’-环单磷硫酸酯、（Rp)-和(Sp)-8-溴- pet -环GMPS）的非对映体对鸟苷3':5'-环单磷酸腺苷（环GMP）-和腺苷3':5'-环单磷酸腺苷（环AMP）依赖性蛋白激酶活性的调节。此外，我们还研究了(Rp)-8-溴- pet -环GMPS对完整人血小板蛋白磷酸化、电场刺激大鼠尾动脉[H-3]-去甲肾上腺素释放和神经源性血管收缩的影响对大鼠尾动脉中表达的纯化的环amp依赖性蛋白激酶（PKG） I α和I β进行动力学分析，发现(Rp)-8-溴- pet -环GMPS是一种竞争性抑制剂，其表观K-i为0.03 μ m。纯化的环amp依赖性蛋白激酶（PKA） II型的活化被拮抗，其表观K-i为10 μ m。（Rp）-8-溴- pet -环GMPS （0.1 mM）拮抗选择性活化剂8-（4-氯苯基硫代）-鸟苷3′：5′-环单磷酸（8- pcpt -环GMP； 0.2 mM）对PKG的活化作用，而不影响(Sp)-5,6-二氯-1- β -d -核呋喃-sylbenzimidazol -3′：5′-环单磷硫代（(Sp)-5,6- dcl -环BiMPS）对PKA的活化；0.1毫米)。4（Rp）-8-溴- pet -环GMP不能被来自牛主动脉的环GMP特异性磷酸二酯酶（PDE） V型水解，但能有效抑制该PDE。5所研究的两种磷硫酸衍生物的相应无硫环核苷酸为8-溴-p - β -苯基- 1，n -2-乙烯鸟苷-3':5'-环单磷酸（8-溴- pet -环GMP）。对电诱导的[H-3]-去甲肾上腺素释放无影响，但浓度依赖性地降低刺激诱导的血管收缩。（Rp）-8-溴- pet -环GMPS （3 μ M）使血管收缩反应向右移位，但不影响刺激引起的氚溢出NO供体3- morpholinosydnon亚胺（SIN-1）用苯肾上腺素（1 μ M）使大鼠尾动脉预收缩。(Rp)-8-溴- pet -环GMPS使SIN-1浓度-舒张曲线平行向右移动，提示舒张是由环GMP/ pkg依赖性机制介导的（Rp）-8-溴- pet -环GMPS对[H-3]-去甲肾上腺素释放增强作用和刺激诱导的福斯克林血管收缩减少作用不受影响。此外，在PKG抑制剂存在的情况下，福斯克林浓度松弛曲线没有改变，这表明在完整细胞中，PKG介导的作用比pka介导的作用具有高选择性结果表明，(Rp)-8-溴- pet -环GMPS是目前最有效和选择性的PKG抑制剂，有助于区分环GMP和环AMP信使通路的激活。因此，这种硫代磷酸酯对映体可能是研究环GMP体外作用的有用工具。
1 The modulation of the guanosine 3':5'-cyclic monophosphate (cyclic GMP)- and adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase activities by the diastereomers of 8-bromo-beta phenyl-1 ,N-2-ethenoguanosine 3',5'-cyclic monophosphorothioate, ((Rp)- and (Sp)-8-bromo-PET-cyclic GMPS) was investigated by use of purified protein kinases. In addition, the effects of (Rp)-8-bromo-PET-cyclic GMPS on protein phosphorylation in intact human platelets and on [H-3]-noradrenaline release and neurogenic vasoconstriction in electrical field stimulated rat tail arteries were also studied.2 Kinetic analysis with purified cyclic GMP-dependent protein kinase (PKG) type I alpha and I beta, which are expressed in the rat tail artery, revealed that (Rp)-8-bromo-PET-cyclic GMPS is a competitive inhibitor with an apparent K-i of 0.03 mu M. The activation of purified cyclic AMP-dependent protein kinase (PKA) type II was antagonized with an apparent K-i of 10 mu M.3 In human platelets, (Rp)-8-bromo-PET-cyclic GMPS (O.1 mM) antagonized the activation of the PKG by the selective activator 8-(4-chlorophenylthio)-guanosine 3':5'-cyclic monophosphate (8-pCPT-cyclic GMP; 0.2 mM) without affecting the activation of PKA by (Sp)-5,6-dichloro-1-beta-D-ribofurano-sylbenzimidazole-3':5'-cyclic monophosphorothioate ((Sp)-5,6-DCl-cyclic BiMPS; 0.1 mM).4 (Rp)-8-bromo-PET-cyclic GMPS was not hydrolysed by the cylcic GMP specific phosphodiesterase (PDE) type V from bovine aorta but potently inhibited this PDE.5 The corresponding sulphur free cyclic nucleotide of the two studied phosphorothioate derivatives, 8-bromo-P beta-phenyl-1 ,N-2-ethenoguanosine-3':5'-cyclic monophosphate (8-bromo-PET-cyclic GMP), had no effect on electrically-induced [H-3]-noradrenaline release but concentration-dependently decreased the stimulation-induced vasoconstriction. (Rp)-8-bromo-PET-cyclic GMPS (3 mu M) shifted the vasoconstriction response to the right without affecting stimulation evoked tritium overflow.6 The NO donor, 3-morpholinosydnonimine (SIN-1) relaxed rat tail arteries precontracted with phenylephrine (1 mu M) The SIN-1 concentration-relaxation curve was shifted in a parallel manner to the right by (Rp)-8-bromo-PET-cyclic GMPS, suggesting that the relaxation was mediated by a cyclic GMP/PKG-dependent mechanism.7 The [H-3]-noradrenaline release-enhancing effect and stimulation-induced decrease in vasoconstriction of forskolin were unaffected by (Rp)-8-bromo-PET-cyclic GMPS. Moreover, the forskolin concentration-relaxation curve was not changed in the presence of the PKG inhibitor, suggesting a high selectivity in intact cells for PKG- over PKA-mediated effects.8 The results obtained indicate that (Rp)-8-bromo-PET-cyclic GMPS presently is the most potent and selective inhibitor of PKG and is helpful in distinguishing between cyclic GMP and cyclic AMP messenger pathways activation. Therefore, this phosphorothioate stereomer may be a useful tool for studying the role of cyclic GMP in vitro.