Inhibition of the ER-Kinase cascade by PD98059 and UO126 counteracts ischemic preconditioning in pig myocardium

Our previous studies suggested a protective role of the extracellular signa l-regulated kinases (ERKs) cascade in ischemic preconditioning (IP) in the porcine heart. To test this hypothesis further, we studied the influence of the novel specific inhibitors of mitogen-activated protein kinase kinases (MEK 1/2) PD98059 (PD) and UO126 (UO) in IP. The substances were infused in tramyocardially and UO also systemically in anesthetized, ventilated, open- chested, male pigs. The local intramyocardial PD and UO infusions occurred before IP and during both reperfusion (RP) phases of IP via four pairs of n eedles (three pairs verum, one solvent) into the risk area (RA). The IP des ign included two cycles of 10-min left anterior descending artery (LAD) occ lusion and 10 min RP, followed by 40 min of occlusion (index ischemia) and of 60 min of RP. Biopsies of the areas of drug infusion were taken after th e second RP cycle of IP. By Western blot analysis, the phosphorylation of E RK 1/2 and of the downstream transcription factor Elk-1 were measured, and the activities of the ERKs were tested by in gel phosphorylation. Only smal l infarcts were detected in the control group animals with the IP period [i nfarct size (IS), infarct area/risk area; IS, 2.5 +/- 0.1%]. Significant we dge-shaped infarcts were seen around the area of the PD and UO infusions. T he effects of PD and UO were concentration dependent. The maximal dose of U O 126 (7.5 mg systemically) was associated with an IS of 65.7 +/- 2.0%. At the end of TP, we observed a significant increase in phospholylation and ac tivities of ERKs. PD (50 mu M) induced a 50% inhibition of ERK-1 and 56% of ERK-2 activities. Phosphorylated ERK-1 and ERK-2 were decreased after micr oinfusion of both PD and UO (50 mu M). Microinfusion of 50 mu M PD also sig nificantly decreased the phosphoryiation of Elk-1 (to 59.2 +/- 8.3% of cont rol conditions). We demonstrate for the first time in vivo that the inhibit ion of ERKs by PD and UO results in a complete cancellation of IF.