Inhibition of cGMP-Dependent Protein Kinase by the Cell-Permeable Peptide DT-2 Reveals a Novel Mechanism of Vasoregulation

Abstract

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 I␣ inhibitor DT-2 on basal and cGMP-stimulated purified recombinant PKG, and compared DT-2 with commonly used PKG inhibitors (8R,9S,11S)-(Ϫ)-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-3Ј,5Ј-cyclic monophosphorothioate (Rp-8-pCPTcGMPS), and (␤-phenyl-1,N2-etheno-8-bromoguanosine-3Ј,5Ј-cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-PETcGMPS). 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.

cGMP-Dependent Protein Kinases and cGMP Phosphodiesterases in Nitric Oxide and cGMP Action

“…Assuming that the PKGI affinities determined in vitro reflect those in the intact cell, it would be predicted that in the absence of compartmentation, these PKGI␤-mediated phosphorylations would occur only at relatively high cGMP levels and after complete activation of PKGI␣. A broad range of PKG sensitivity to cGMP is supported by studies of PKG function in cerebral arteries using DT-2, a specific PKG peptide inhibitor (Dostmann et al, 2000;Taylor et al, 2004). Even in the absence of agents to increase cGMP, DT-2 treatment increases basal tone consistent with blockage of a low level of PKG activity.…”

“…Some cN analogs (e.g., 1,N 2 -phenyletheno-cGMP) are more potent PKGI activators than cGMP, and others, (e.g., 8-bromo-␤-phenyl-1,N 2 -ethenoguanosine-3Ј,5Ј-cyclic monophosphorothioate, Rp-isomer) bind to the allosteric site but only partially activate catalysis; the latter compound is commonly used as a PKGI inhibitor because it blocks access of the more effective activator, cGMP, to the binding sites (Sekhar et al, 1992;Butt et al, 1994b;Taylor et al, 2004;Poppe et al, 2008;Valtcheva et al, 2009). Cyclic nucleotide analogs have proved to be highly useful tools in investigating biological effects mediated by PKAs, EPACs, or PKGs; the analogs freely traverse the cell membrane to directly act on the target proteins, thereby circumventing involvement of proteins that generate the signaling molecule, factors involved in delivery of the signal to the target cell, specific membrane receptors, or the adenylyl or guanylyl cyclases that produce the cNs (Beebe et al, 1988a,b;Francis et al, 1988;Christensen et al, 2003;Dao et al, 2006;Poppe et al, 2008).…”

Blockade of the purinergic P2Y 12 receptor greatly increases the platelet inhibitory actions of nitric oxide

“…This may indicate that the inhibition of platelet aggregation occurs through a cGK-independent mechanism. Alternatively, and possibly more likely, this may reflect the limitations of these experimental compounds (27)(28)(29).…”

Adipocyte-Specific Mineralocorticoid Receptor Overexpression in Mice Is Associated With Metabolic Syndrome and Vascular Dysfunction: Role of Redox-Sensitive PKG-1 and Rho Kinase

“…The inhibitors DT-3 and Rp-8-Br-PET-cGMPS used in our studies are both highly potent, permeable, and selective for PKG-1a, and reduced cyclic guanosine monophosphate (cGMP)-stimulated PKG activity (23).…”