Highly specific, membrane-permeant peptide blockers of cGMP-dependent protein kinase I inhibit NO-induced cerebral dilation

ArticleinProceedings of the National Academy of Sciences 97(26):14772-7 · January 2001with13 Reads
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.
    • "A full description can be found in the Supplemental Experimental Procedures. Kinetic Analysis Activity of recombinant PKG Ia toward the synthetic substrate peptide (W15, TQAKRKKSLAMA) was measured by [g-32 P]ATP incorporation assay as described by Dostmann et al. (2000) with some modifications. In brief, reactions were initiated when 0.1 mM [g-32 P] ATP (200–300 cpm/pmol) was incubated with vials preincubated with 50 mM MES (pH 6.9), 1 mM Mg acetate, 10 mM NaCl, 10 mM DTT, 1 mg/ml BSA, 10 mM W15 substrate, 1 nM PKG Ia, and 10 ml of 103 S-tide stocks, in a 100-ml reaction volume at 30 C. Prior (A) Diameter measurements for endothelium-denuded, reversibly permeabilized arteries demonstrating myogenic tone response in (Aa) control and (Ab) S1.5- treated arteries. "
    [Show abstract] [Hide abstract] ABSTRACT: PKG is a multifaceted signaling molecule and potential pharmaceutical target due to its role in smooth muscle function. A helix identified in the structure of the regulatory domain of PKG Iα suggests a novel architecture of the holoenzyme. In this study, a set of synthetic peptides (S-tides), derived from this helix was found to bind to and activate PKG Iα in a cGMP-independent manner. The most potent S-tide derivative (S1.5) increased the open probability (NPo) of the potassium channel KCa1.1 to levels equivalent to saturating cGMP. Introduction of S1.5 to smooth muscle cells in isolated, endothelium-denuded cerebral arteries through a modified reversible permeabilization (RP) procedure inhibited myogenic constriction. In contrast, in endothelium-intact vessels, S1.5 had no effect on myogenic tone. This suggests that PKG Iα activation by S1.5 in vascular smooth muscle would be sufficient to inhibit augmented arterial contractility that frequently occurs following endothelial damage associated with cardiovascular disease.
    Full-text · Article · Dec 2015
    • "Even though an inhibition of PKG would probably lead to severe side effects in prolonged treatment it could be used to antagonize the pathologic hypotonic condition (vasoplegia) encountered during an anaphylactic/septic shock. The efficacy of the PKG inhibitors DT-2 and DT-3 in decreasing NO mediated vasodilation has been demonstrated in isolated cerebral arteries [30,44]. Furthermore investigations on the regulation of PKG-I expression levels revealed a cGMP dependent ubiquitination and degradation of PKG-I in cultured vascular smooth muscle cells. "
    [Show abstract] [Hide abstract] ABSTRACT: cGMP-dependent protein kinases (PKG) exhibit diverse physiological functions in the mammalian system e.g., in vascular and gastrointestinal smooth muscles, in platelets, in kidney, in bone growth, nociception and in the central nervous system. Furthermore, PKG were found in insects and in the malaria parasite Plasmodium falciparum. Two different genes of PKG exist: a) the PKG-I gene that is expressed as cytosolic PKG-Iα or PKG-Iβ isoform, and b) the PKG-II gene, which expresses the membrane associated PKG-II protein. The enzyme kinetics, the localization and the substrates of these PKG enzymes differ utilizing different physiological functions. Various inhibitors of PKG were developed directed against diverse functional regions of the kinase. These inhibitors of PKG have been used to analyse the specific functions of these enzymes. The review article will summarize these different inhibitors regarding their specificity and their present applications in vitro and in vivo. Furthermore, it will be discussed that the distinct inhibition of the PKG enzymes could be used as a valuable pharmacological target e.g., in the treatment of cardiovascular diseases, diarrhea, cancer or malaria.
    Full-text · Article · Feb 2013
    • "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.. "
    [Show abstract] [Hide abstract] 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.
    Full-text · Article · Dec 2012
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