These data suggest that b adrenergic stimulation is just partially in charge of PKA activation during TP protocol and that another pathway, perhaps ROS mediated,14 may also be involved. Certainly, we have shown previously that the free radical scavenger N glycine applied all through pre ischaemia buy PF299804 abolishes cardioprotection by TP since it does for IP. 2 Stowe, Moreover and colleagues15 have reported that hypothermia averagely increases superoxide concentration in myocardium. Interestingly, it’s been shown by others that b adrenergic stimulation can be viewed as a trigger of IP4 and that recurring stimulation with norepinephrine or isoproterenol mimics IP. Proposed mechanisms for cardioprotection by b adrenergic activation of PKA include attenuation of calpainmediated degradation pathways and b adrenergic desensitization4. A major problem with Immune system applying protein kinase inhibitors to dissect signal transduction pathways is their insufficient specificity. It could hinder other kinases including Akt, 17 Although H 89 is a strong PKA inhibitor. We were unable to detect any change in phosphorylation of either protein following a TP protocol and the Akt GSK3 pathway has been implicated in cardioprotection by IP,18 although our own data3 and that of other19 have questioned the central purpose of this pathway prior to ischaemia. Hausenloy et al. showed Ip Address induced Akt phosphorylation at 15 min of reperfusion following continuous ischaemia, but we were also unable to detect any changes in Akt or GSK3 phosphorylation by TP at 15 min reperfusion. However, we can not completely because phosphorylation could be transient, though in studies where Akt activation and GSK3 inhibition were found buy Gemcitabine to be important for cardioprotection, phosphorylation of these kinases was very steady and substantial during reperfusion and preischaemia exclude involvement with this pathway in TP. Thus, our data do not support an important role of Akt and GSK3 phosphorylation in the TP signalling process. Consecutive PKA and PKC activation throughout TP and pharmacologically induced straight PKA/PKC activation We’ve previously found that PKC activation is crucial for TP mediated cardioprotection,2 and here, we demonstrate that the PKA inhibitor H 89, which itself has little impact on PKC activity,17 can prevent both this PKC activation and cardioprotection, implying that PKA activation is upstream of PKC activation in the TP signalling pathway. Our data further support this finding. Ergo, treatment of rat hearts repeatedly using the w adrenergic agonist isoproterenol and then adenosine, to activate PKC, triggered acutely potent cardioprotection that substantially exceeded the protection afforded by either agent alone or added simultaneously and allowed hearts to recuperate completely after 30-min normothermic worldwide ischaemia.