Inhibition of cGMP-dependent protein kinase by the cell-permeable peptide DT-2 reveals a novel mechanism of vasoregulation.
Cyclic GMP-dependent protein kinase (PKG) serves as an important physiological regulator of vascular reactivity and tone. However, available inhibitors of PKG have exhibited variable effects in intact tissue, hindering the elucidation of the functional role of PKG in blood vessels. In this study, we have determined the effects of our previously engineered potent and selective PKG Ialpha inhibitor DT-2 on basal and cGMP-stimulated purified recombinant PKG, and compared DT-2 with commonly used PKG inhibitors (8R,9S,11S)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cycloocta-(c,d,e)-trinden-1-one (KT-5823), Rp-8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate (Rp-8-pCPT-cGMPS), and (beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-PET-cGMPS). As expected, all inhibitors reduced cGMP-stimulated PKG activity. However, only DT-2 decreased cGMP-independent or basal PKG activity, whereas KT5823 showed no effect and the Rp-compounds actually had partial agonist activity. To evaluate the potential functional impact of this unique inhibition by DT-2 under physiologically relevant conditions, we analyzed the inhibitors in isolated pressurized cerebral arteries. KT-5823 and Rp-8-pCPT-cGMPS demonstrated marginal reversal of vasodilation induced by 8-Br-cGMP. By comparison, DT-2 completely reversed 8-Br-cGMP induced dilations with comparable potency to Rp-8-Br-PET-cGMPS. In fact, DT-2 constricted arteries beyond their starting (pre-8-Br-cGMP) diameters and caused constriction even in the absence of exogenous 8-Br-cGMP, an effect that was not observed with any other inhibitor. The direct constricting effect of DT-2 was essentially abolished in cultured arteries, where PKG expression was reduced by approximately 90%. These findings indicate that DT-2 not only effectively inhibits cGMP-stimulated PKG activity but also reduces basal PKG activity both in vitro and in vivo. Moreover, these distinctive inhibitory properties of DT-2 suggest an important role for constitutive PKG activity in the continuous regulation of cerebral artery tone.
Taylor
Mark S
MS
University of Vermont, Department of Pharmacology, College of Medicine, Burlington 05405-0075, USA.
Okwuchukwuasanya
Chris
C
Nickl
Christian K
CK
Tegge
Werner
W
Brayden
Joseph E
JE
Dostmann
Wolfgang R G
WR
D000818:Animals; D002227:Carbazoles / Q000494:pharmacology; D002536:Cerebral Arteries / Q000187:drug effects; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D066298:In Vitro Techniques; D007211:Indoles / Q000494:pharmacology; D008297:Male; D051381:Rats; D013873:Thionucleotides / Q000494:pharmacology; D014661:Vasoconstriction / Q000187:drug effects; D014664:Vasodilation / Q000187:drug effects
C090860:8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphorothioate; D002227:Carbazoles; D004791:Enzyme Inhibitors; D007211:Indoles; D013873:Thionucleotides; C073601:KT 5823; C016276:8-bromocyclic GMP; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP