Abstract
Small-molecule modulators of cGMP signaling are of interest to basic and clinical research. The cGMP-dependent protein kinase type I (cGKI) is presumably a major mediator of cGMP effects, and the cGMP analogue Rp-8-Br-PET-cGMPS (Rp-PET) (chemical name: beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer) is currently considered one of the most permeable, selective, and potent cGKI inhibitors available for intact cell studies. Here, we have evaluated the properties of Rp-PET using cGKI-expressing and cGKI-deficient primary vascular smooth muscle cells (VSMCs), purified cGKI isozymes, and an engineered cGMP sensor protein. cGKI activity in intact VSMCs was monitored by cGMP/cGKI-stimulated cell growth and phosphorylation of vasodilator-stimulated phosphoprotein. Unexpectedly, Rp-PET (100 microm) did not efficiently antagonize activation of cGKI by the agonist 8-Br-cGMP (100 microm) in intact VSMCs. Moreover, in the absence of 8-Br-cGMP, Rp-PET (100 microm) stimulated cell growth in a cGKIalpha-dependent manner. Kinase assays with purified cGKI isozymes confirmed the previously reported inhibition of the cGMP-stimulated enzyme by Rp-PET in vitro. However, in the absence of the agonist cGMP, Rp-PET partially activated the cGKIalpha isoform. Experiments with a fluorescence resonance energy transfer-based construct harboring the cGMP binding sites of cGKI suggested that binding of Rp-PET induces a conformational change similar to the agonist cGMP. Together, these findings indicate that Rp-PET is a partial cGKIalpha agonist that under certain conditions stimulates rather than inhibits cGKI activity in vitro and in intact cells. Data obtained with Rp-PET as cGKI inhibitor should be interpreted with caution and not be used as sole evidence to dissect the role of cGKI in signaling processes.
Highlights
Based on pharmacological and genetic studies, the cGK type I is considered the major mediator of cGMP signaling in many tissues including the cardiovascular system [5,6,7,8]
Opposing effects of cGMP/cGK type I (cGKI) signaling have been reported on the growth and phenotype of vascular smooth muscle cells (VSMCs) [11, 12]
It was tested whether Rp-PET could inhibit tial agonistic effect of Rp-PET observed in intact cells and sugcGMP/cGKI-stimulated VSMC growth and phosphorylation gested that Rp-PET might preferentially activate the cGKI␣
Summary
Based on pharmacological and genetic studies, the cGK type I (cGKI) is considered the major mediator of cGMP signaling in many tissues including the cardiovascular system [5,6,7,8]. We intended to validate the selectivity and efficacy of Rp-PET as a cGKI inhibitor in intact cells by comparing its effects on cGKI-expressing and cGKI-deficient VSMCs. Surprisingly, Rp-PET did not efficiently inhibit but rather stimulated cGKI-mediated processes in VSMCs. In vitro experiments with purified cGKI isozymes and an engineered cGKI-based cGMP sensor protein supported these findings, suggesting that Rp-PET is a partial agonist rather than an antagonist of cGKI␣.
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