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1 Department of Physiology, University of South Alabama, Mobile, AL, USA; Department of Cardiology, Ernst-Moritz-Arndt Universitat, Greifswald, Germany
2 Department of Physiology, University of South Alabama, Mobile, AL, USA
3 Department of Pharmacology, University of Vermont, Burlington, VT, USA
4 Department of Physiology, University of South Alabama, Mobile, AL, USA; Department of Medicine, University of South Alabama, Mobile, AL, USA
* To whom correspondence should be addressed. E-mail: mcohen{at}usouthal.edu.
Bradykinin and acetylcholine (ACh) trigger preconditioning by KATP channel-dependent production of reactive oxygen species (ROS). Recent evidence suggests that ROS production may in turn be influenced by cyclic GMP-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 minutes in reduced MitoTracker Red which becomes fluorescent only after exposure to ROS. Bradykinin (400nM) and ACh (250µM) caused a 49.9±5.9% and 46.8±1.7% increase in ROS production, resp. (p< 0.005 vs untreated cells). Co-incubation with DT-3 (250nM) abolished both the ACh- and bradykinin-induced ROS signal, while a non-permeable form of the peptide (W45) had no effect on ACh-induced ROS production. DT-3 was unable to block ROS production from diazoxide (100µM), a selective opener of mitochondrial KATP channels, suggesting that these channels are downstream of PKG. DT-2 (125nM) also prevented ACh from triggering ROS production. 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (100µM), a cyclic GMP 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 bradykinin's anti-infarct effect 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. While 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.
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