cGMP
Bradykinin mimics ischemic preconditioning by
generating reactive oxygen species (ROS). To identify intermediate steps
leading to ROS generation, rabbit cardiomyocytes were incubated in reduced MitoTracker
Red that becomes fluorescent after exposure to ROS. Fluorescence intensity in
treated cells was expressed as % of that in paired untreated cells. Bradykinin
(500nM) caused 51+/-16% increase in ROS generation (p<0.001). Co-incubation
with either bradykinin B2 receptor blocker HOE140 (5 micro M) or free radical
scavenger N-(2-mercaptopropionyl) glycine (1mM) prevented this increase
confirming the response was receptor-mediated and ROS were actually being
measured. Closing mitochondrial KATP (mKATP)channels with 5-hydroxydecanoate
(5HD, 1mM) prevented increased ROS generation. Bradykinin-induced ROS
generation was blocked by L-NAME (200 micro M) implicating nitric oxide as an
intermediate. Blockade of guanylyl cyclase with ODQ (10 micro M) aborted bradykinin-induced
ROS generation, but not that from diazoxide, a direct opener of mKATP channels.
The PKG blocker 8-Br-cGMPS (25 micro M) eliminated bradykinin's effect.
Conversely, direct activation of PKG with 8-pCPT-cGMP (100 micro M) increased
ROS generation (39+/-15%, p<0.004) similar to bradykinin. This increase was
blocked by 5HD. Finally, the nitric oxide donor SNAP (1 micro M)increased ROS
by 34+/-6%. This increase was also blocked by 5HD. In intact rabbit hearts
bradykinin (400nM) decreased infarction from 30.5+/-3.0% of the risk zone in
control hearts to 11.9+/-1.4% (p<0.01). This protection was aborted by
either L-NAME (200 micro M) or ODQ (2 micro M)(35.4+/-5.7% and 30.4+/-3.0%
infarction, resp., pNS vs control). Hence, bradykinin preconditions through
receptor-mediated production of nitric oxide which activates guanylyl cyclase.
The resulting cGMP activates PKG that opens mKATP. Subsequent release of ROS
triggers cardioprotection.