| Ischemic preconditioning (IPC) refers to the phenomenon by which brief episodes of ischemia and reperfusion render the myocardium resistant to necrosis from a subsequent otherwise lethal ischemia. IPC is triggered by receptors. Cardioprotection from acetylcholine (ACh), bradykinin (BK) and opioids is dependent on reactive oxygen species (ROS), which are thought to result from opening of mitochondrial ATP-sensitive potassium channels (mKATP). The aim of this dissertation was to determine the signaling components between cell membrane receptors and opening of mKATP.; Infarct size was measured in buffer-perfused isolated rabbit hearts. Myocardial infarction was induced by 30 min of regional ischemia with reperfusion. IPC was induced by 5 min global ischemia and 10 min reperfusion prior to the 30 min regional ischemia. ROS were measured in isolated adult rabbit cardiomyocytes. Phosphorylation of proteins or cGMP was measured in myocardial biopsies.; Because inhibition of respiratory chain complex III blocked ACh's cardioprotection, while NAD(P)H blockade did not, I conclude that mitochondria are the source of the ROS burst. My studies reveal that ACh requires phosphatidylinositol-3-OH-kinase (PI3-kinase) and at least one tyrosine kinase to trigger the preconditioned state while adenosine (Aden) does not. My experiments also show that the epidermal growth factor receptor (EGFR) is likely to be the site of the tyrosine kinase. In other cell types ACh causes release of HB-EGF by activating a metalloproteinase. As expected a metalloproteinase inhibitor blocked ACh's protection.; My experiments reveal that ACh triggers mKATP opening by activating nitric oxide synthase (NOS). Nitric oxide activates guanylyl cyclase (GC) to generate cGMP, which activates protein kinase G (PKG). ACh's protection was blocked by either L-NIO (a NOS blocker) or 1H-[1, 2, 4]oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ, a GC blocker). Similarly, cardioprotection from 8-(4-chlorophenylthio) guanosine 3',5' -cyclic monophosphate (8-pCPT-cGMP, a direct activator of PKG) was blocked by a free radical scavenger and an mKATP blocker.; In conclusion, ACh protects the heart by a pathway different from that used by Aden. EGFR, PI3-kinase, NOS, GC, mKATP, and ROS, in that order, are essential signaling components by which ACh triggers preconditioning. Aden bypasses all of the above to protect the heart. |
