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Peptide blockers of PKG inhibit ROS generation by acetylcholine and bradykinin in cardiomyocytes but fail to block protection in the whole heart

Departments of ¹Physiology and ²Medicine, University of South Alabama College of Medicine, Mobile, Alabama; ³Department of Cardiology, Ernst-Moritz-Arndt Universität, Greifswald, Germany; and ⁴Department of Pharmacology, University of Vermont, Burlington, Vermont

Submitted 26 August 2004 ; accepted in final form 2 December 2004

Bradykinin and acetylcholine (ACh) trigger preconditioning by ATP-sensitive K⁺ (K_ATP) channel-dependent production of reactive oxygen species (ROS). Recent evidence suggests that ROS production may in turn be influenced by cGMP-dependent protein kinase (PKG). This study utilized DT-2 and DT-3 peptides, highly specific membrane-permeable blockers of PKG. Rabbit cardiomyocytes were incubated for 15 min in reduced MitoTracker red, which becomes fluorescent only after exposure to ROS. Bradykinin (400 nM) and ACh (250 µM) caused a 49.9 ± 5.9% and 46.8 ± 1.7% increase in ROS production, respectively (P < 0.005 vs. untreated cells). Coincubation with DT-3 (250 nM) abolished both the ACh- and bradykinin-induced ROS signal, whereas a nonpermeable form of the peptide (W45) had no effect on ACh-induced ROS production. DT-3 was unable to block ROS production from diazoxide (100 µM), a selective opener of mitochondrial K_ATP channels, suggesting that these channels are downstream of PKG. DT-2 (125 nM) also prevented ACh from triggering ROS production. 8-(4-Chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (100 µM), a cGMP analog and potent direct activator of PKG, increased ROS production of cardiomyocytes by 44.7 ± 7.1% (P < 0.001 vs. untreated cells). This increase was blocked by DT-2. Neither DT-2 nor DT-3 could block the anti-infarct effect of bradykinin in isolated rabbit hearts. Studies with fluorescent-tagged DT-3 revealed that it was confined to endothelial cells and never reached the myocytes. We conclude that both bradykinin and ACh trigger ROS generation by a pathway that includes PKG. Although the peptides may be inappropriate for a whole heart model, they are likely to become important tool drugs for elucidation of signal transduction pathways in cell preparations.

protein kinase G; reactive oxygen species

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