Many signal transduction pathways are mediated by the second messengers cGMP and cAMP, cGMP- and cAMP-dependent protein kinases (cGK and PKA), phosphodiesterases, and ion channels. To distinguish among the different cGMP effectors, inhibitors of cGK and PKA have been developed including the K-252 compound KT5823 and the isoquinolinesulfonamide H89. KT5823, an in vitro inhibitor of cGK, has also been used in numerous studies with intact cells to implicate or rule out the involvement of this protein kinase in a given cellular response. However, the efficacy and specificity of KT5823 as cGK inhibitor in intact cells or tissues have never been demonstrated. Here, we analyzed the effects of both KT5823 and H89 on cyclic-nucleotide-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in intact human platelets and rat mesangial cells. These two cell types both express high levels of cGK. KT5823 inhibited purified cGK. However, with both intact human platelets and rat mesangial cells, KT5823 failed to inhibit cGK-mediated serine 157 and serine 239 phosphorylation of VASP induced by nitric oxide, atrial natriuretic peptide, or the membrane-permeant cGMP analog, 8-pCPT-cGMP. KT5823 enhanced 8-pCPT-cGMP-stimulated VASP phosphorylation in platelets and did not inhibit forskolin-stimulated VASP phosphorylation in either platelets or mesangial cells. In contrast H89, an inhibitor of both PKA and cGK, clearly inhibited 8-pCPT-cGMP and forskolin-stimulated VASP phosphorylation in the two cell types. The data indicate that KT5823 inhibits purified cGK but does not affect a cGK-mediated response in the two different cell types expressing cGK I. These observations indicate that data that interpret the effects of KT5823 in intact cells as the major or only criteria supporting the involvement of cGK clearly need to be reconsidered.

中文翻译：

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KT5823在体外可抑制cGMP依赖性蛋白激酶活性，但在完整的人血小板和大鼠肾小球系膜细胞中则不会。

第二信使cGMP和cAMP，依赖cGMP和cAMP的蛋白激酶（cGK和PKA），磷酸二酯酶和离子通道介导许多信号转导途径。为了区分不同的cGMP效应子，已经开发了cGK和PKA抑制剂，包括K-252化合物KT5823和异喹啉磺酰胺H89。KT5823，一种体外cGK抑制剂，也已用于完整细胞的众多研究中，以暗示或排除该蛋白激酶参与给定的细胞反应。但是，尚未证明KT5823作为cGK抑制剂在完整细胞或组织中的功效和特异性。在这里，我们分析了完整人类血小板和大鼠肾小球系膜细胞中，KT5823和H89对环核苷酸介导的血管舒张剂刺激的磷蛋白（VASP）磷酸化的影响。这两种细胞类型均表达高水平的cGK。KT5823抑制纯化的cGK。但是，对于完整的人血小板和大鼠肾小球系膜细胞，KT5823均无法抑制由一氧化氮，心钠素或膜渗透性cGMP类似物8-pCPT-cGMP诱导的cGK介导的VASP的丝氨酸157和丝氨酸239磷酸化。KT5823增强了血小板中8-pCPT-cGMP刺激的VASP磷酸化，并且在血小板或肾小球膜细胞中均未抑制福斯高林刺激的VASP磷酸化。相比之下，PKA和cGK的抑制剂H89明显抑制了两种细胞类型中的8-pCPT-cGMP和福司柯林刺激的VASP磷酸化。数据表明，KT5823在表达cGK I的两种不同细胞类型中抑制纯化的cGK，但不影响cGK介导的反应。