Exploring the mechanisms of vascular smooth muscle tone with highly specific, membrane-permeable inhibitors of cyclic GMP-dependent protein kinase Ialpha.
The structural similarity of cyclic GMP-dependent protein kinase (cGPK) and cyclic AMP-dependent protein kinase (cAPK) has made it difficult to study cGPK pathways independent of those mediated by cAPK, primarily due to the lack of potent and selective cGPK inhibitors. We recently reported a novel peptide library screen specifically designed to select for tight-binding peptides that identified selective inhibitors of cGPK [Proc Natl Acad Sci USA, 97 (2000) 14772]. Iterative deconvolution of octameric library arrays on paper identified the sequence LRK(5)H (W45). 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 cAPK, respectively. Cellular internalization of highly charged W45 was accomplished by N-terminal fusion of membrane translocation sequences from either the human immunodeficiency virus tyrosine aminotransferase protein (47-59) DT-2 or from the Drosophila Antennapedia homeodomain (43-58) DT-3, respectively. For both fusion peptides, DT-2 and DT-3, we observed a potentiating effect with respect to the inhibitory potency, with K(i) values 40- to 80-fold lower than W45. Fluorescein-labeled DT-2 and DT-3 demonstrated rapid translocation through the cytosol and nuclei in a time-dependent manner using cultured cells and intact tissue samples (cerebral arteries). The physiological effects of DT-2 and DT-3 as selective cGPK inhibitors in smooth muscle were studied in small intact arteries. Nitric oxide, a cyclic GMP/cGPK activator, elicited a concentration-dependent dilation of isolated rat cerebral arteries, which was markedly inhibited by DT-2 and DT-3. Collectively, these results indicate that DT-2 and DT-3 effectively inhibit nitric oxide-induced vasodilation, further emphasizing the central role for cGPK in the modulation of vascular contractility.
Department of Pharmacology, University of Vermont, College of Medicine, Given Building, 89 Beaumont Avenue, Burlington, VT 05405, USA. wolfgang.dostmann@uvm.edu
Wolfgang R G
WR
Dostmann
Werner
W
Tegge
Ronald
R
Frank
Christian K
CK
Nickl
Mark S
MS
Taylor
Joseph E
JE
Brayden
D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000138:chemical synthesis; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology*; D019151:Peptide Library; D051381:Rats; D015398:Signal Transduction
D019151:Peptide Library; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases