Dostmann, W.R. et al. Highly specific, membrane-permeant peptide blockers of cGMP-dependent protein kinase I inhibit NO-induced cerebral dilation. Proc. Natl. Acad. Sci. USA 97, 14772-14777

Department of Pharmacology, Department of Molecular Physiology and Biophysics, University of Vermont, College of Medicine, Burlington, VT 05405-0068, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 01/2001; 97(26):14772-7. DOI: 10.1073/pnas.97.26.14772
Source: PubMed


Arrays of octameric peptide libraries on cellulose paper were screened by using (32)P-autophosphorylated cGMP-dependent protein kinase Ialpha (cGPK) to identify peptide sequences with high binding affinity for cGPK. Iterative deconvolution of every amino acid position in the peptides identified the sequence LRK(5)H (W45) as having the highest binding affinity. Binding of W45 to cGPK resulted in selective inhibition of the kinase with K(i) values of 0.8 microM and 560 microM for cGPK and cAMP-dependent protein kinase (cAPK), respectively. Fusion of W45 to membrane translocation signals from HIV-1 tat protein (YGRKKRRQRRRPP-LRK(5)H, DT-2) or Drosophila Antennapedia homeo-domain (RQIKIWFQNRRMKWKK-LRK(5)H, DT-3) proved to be an efficient method for intracellular delivery of these highly charged peptides. Rapid translocation of the peptides into intact cerebral arteries was demonstrated by using fluorescein-labeled DT-2 and DT-3. The inhibitory potency of the fusion peptides was even greater than that for W45, with K(i) values of 12.5 nM and 25 nM for DT-2 and DT-3, respectively. Both peptides were still poor inhibitors of cAPK. Selective inhibition of cGPK by DT-2 or DT-3 in the presence of cAPK was demonstrated in vitro. In pressurized cerebral arteries, DT-2 and DT-3 substantially decreased NO-induced dilation. This study provides functional characterization of a class of selective cGPK inhibitor peptides in vascular smooth muscle and reveals a central role for cGPK in the modulation of vascular contractility.

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    • "To investigate the contribution of PKG to NaHS-induced vasodilation, vessel rings were pre-treated with DT-2 [34] prior to exposure to NaHS. Such pre-treatment attenuated the vasorelaxation brought about by NaHS, indicating that NaHS-induced relaxation is PKG-I-dependent.. "
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    ABSTRACT: A growing body of evidence suggests that hydrogen sulfide (H(2)S) is a signaling molecule in mammalian cells. In the cardiovascular system, H(2)S enhances vasodilation and angiogenesis. H(2)S-induced vasodilation is hypothesized to occur through ATP-sensitive potassium channels (K(ATP)); however, we recently demonstrated that it also increases cGMP levels in tissues. Herein, we studied the involvement of cGMP-dependent protein kinase-I in H(2)S-induced vasorelaxation. The effect of H(2)S on vessel tone was studied in phenylephrine-contracted aortic rings with or without endothelium. cGMP levels were determined in cultured cells or isolated vessel by enzyme immunoassay. Pretreatment of aortic rings with sildenafil attenuated NaHS-induced relaxation, confirming previous findings that H(2)S is a phosphodiesterase inhibitor. In addition, vascular tissue levels of cGMP in cystathionine gamma lyase knockouts were lower than those in wild-type control mice. Treatment of aortic rings with NaHS, a fast releasing H(2)S donor, enhanced phosphorylation of vasodilator-stimulated phosphoprotein in a time-dependent manner, suggesting that cGMP-dependent protein kinase (PKG) is activated after exposure to H(2)S. Incubation of aortic rings with a PKG-I inhibitor (DT-2) attenuated NaHS-stimulated relaxation. Interestingly, vasodilatory responses to a slowly releasing H(2)S donor (GYY 4137) were unaffected by DT-2, suggesting that this donor dilates mouse aorta through PKG-independent pathways. Dilatory responses to NaHS and L-cysteine (a substrate for H(2)S production) were reduced in vessels of PKG-I knockout mice (PKG-I-/-). Moreover, glibenclamide inhibited NaHS-induced vasorelaxation in vessels from wild-type animals, but not PKG-I-/-, suggesting that there is a cross-talk between K(ATP) and PKG. Our results confirm the role of cGMP in the vascular responses to NaHS and demonstrate that genetic deletion of PKG-I attenuates NaHS and L-cysteine-stimulated vasodilation.
    PLoS ONE 12/2012; 7(12):e53319. DOI:10.1371/journal.pone.0053319 · 3.23 Impact Factor
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    • "As sGC and PDE5 have already been implicated as key cGMP regulators (Figure 4), the involvement of phospho-PDE5 (P-PDE5) was scrutinized in regards to the maintenance of VSM [cGMP]i. To block the phosphorylation of PDE5, we applied DT-2, a membrane-permeable and specific inhibitor of PKG1α (Dostmann et al., 2000; Nickl et al., 2010). NO-induced cGMP transients (5 nM NO pulse, MAHMA/NO) elicited in the presence of DT-2 were markedly broader (~20–30% increase as measured by peak width and area under the curve; Figure 5A; Table 2), attributing the phosphorylated PDE5 as a key manager of VSM [cGMP]i. "
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    ABSTRACT: Nitric oxide (NO) is a potent dilator of vascular smooth muscle (VSM) by modulating intracellular cGMP ([cGMP](i)) through the binding and activation of receptor guanylyl cylases (sGC). The kinetic relationship of NO and sGC, as well as the subsequent regulation of [cGMP](i) and its effects on blood vessel vasodilation, is largely unknown. In isolated VSM cells exposed to both pulsed and clamped NO we observed transient and sustained increases in [cGMP](i), with sub-nanomolar sensitivity to NO (EC(50) = 0.28 nM). Through the use of pharmacological inhibitors of sGC, PDE5, and PKG, a comprehensive VSM-specific modeling algorithm was constructed to elucidate the concerted activity profiles of sGC, PDE5, phosphorylated PDE5, and PDE1 in the maintenance of [cGMP](i). In small pressure-constricted arteries of the resistance vasculature we again observed both transient and sustained relaxations upon delivery of pulsed and clamped NO, while maintaining a similarly high sensitivity to NO (EC(50) = 0.42 nM). Our results propose an intricate dependency of the messengers and enzymes involved in cGMP homeostasis, and vasodilation in VSM. Particularly, the high sensitivity of sGC to NO in primary tissue indicates how small changes in the concentrations of NO, irrespective of the form of NO delivery, can have significant effects on the dynamic regulation of vascular tone.
    Frontiers in Pharmacology 07/2012; 3:130. DOI:10.3389/fphar.2012.00130 · 3.80 Impact Factor
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    • "These results suggested that the binding of MYPT1 to PKG1α is not mediated by a LZ-LZ interaction. Because PKG1α prefers binding to RR and RK motifs (Dostmann et al., 2000) and there is an RK motif in the aa 888–928 sequence of MYPT1, the relevance of this sequence was investigated (Given et al., 2007). Mutants were generated, which lack or contain this sequence and which also lack or contain the leucine zipper. "

    Affinity Chromatography, 03/2012; , ISBN: 978-953-51-0325-7
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