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.
