Sebastian Philipp

Postconditioning’s protection is not dependent on circulating blood factors or cells but involves adenosine receptors and requires PI3–kinase and guanylyl cyclase activation

Abstract Protection from postconditioning has been documented in in situ animal models and it has been proposed that it is targeting circulating leukocytes. We therefore tested whether postconditioning can protect leukocytefree, buffer–perfused rabbit hearts. Infarct size was measured with triphenyltetrazolium staining. In control hearts undergoing 30 min of regional ischemia and 2 h of reperfusion, 33.3 ± 2.2% of the risk zone infarcted. The protocol previously used in open–chest animals of four postconditioning cycles of 30 s reperfusion/30 s ischemia starting at the beginning of reperfusion decreased infarction to only 24.8 ± 2.5% of the risk zone in these isolated hearts. Because of the meager protection induced by four 30 s postconditioning cycles, we evaluated the effect of postconditioning with 6 cycles of 10 s reperfusion/10 s ischemia starting at the beginning of reperfusion. Robust salvage was seen with only 10.4 ± 3.4% of the risk zone infarcting (p < 0.001 vs control and p < 0.003 vs 4 cycles of 30 s ischemia). The 10s protocol was used in all studies of signal transduction. Wortmannin (100 nM), a phosphatidylinositol 3– (PI3–) kinase antagonist, infused for 20 min starting 5 min before reperfusion, blocked postconditioning’s, protection (31.2 ± 4.2% infarction) as did 1H–[1,2,4]oxadiazole[4,3–a]quinoxalin–1–one (ODQ) (2 µM) a guanylyl cyclase inhibitor (36.9 ± 5.3%) and 8–p–(sulfophenyl) theophylline (SPT) (100 µM), a non–specific adenosine receptor blocker (34.2 ± 2.8%). Thus, postconditioning’s protection is not dependent on circulating blood factors or cells, and its anti–infarct effect appears to require PI3–kinase activation, stimulation of guanylyl cyclase and occupancy of adenosine receptors. These signaling steps have also been identified in preconditioning and during pharmacologic cardioprotection and suggest commonality of a protective mechanism.

Localizing extracellular signal–regulated kinase (ERK) in pharmacological preconditioning's trigger pathway

AbstractAcetylcholine (ACh) and opioid receptor agonists trigger the preconditioned phenotype through sequential activation of the epidermal growth factor (EGF) receptor, phosphatidylinositol 3-kinase (PI3-K), Akt, and nitric oxide synthase (NOS), and opening of mitochondrial (mito) KATP channels with the generation of reactive oxygen species (ROS). Although extracellular signal–regulated kinase (ERK) has recently been reported to be part of this pathway, its location has not been determined. To address this issue, we administered a 5–min pulse of ACh (550 µM) prior to 30 min of ischemia in isolated rabbit hearts. It reduced infarction from 30.4 ± 2.2% of the risk zone in control hearts to 12.3 ± 2.8% and co–administration of the MEK, and, therefore, downstream ERK inhibitor U0126 abolished protection (29.1 ± 4.6% infarction) con.rming ERKs involvement. MitoKATP opening was monitored in adult rabbit cardiomyocytes by measuring ROS production with MitoTracker Red. ROS production was increased by each of three G protein–coupled agonists: ACh (250 µM), bradykinin (BK) (500 nM), and the δ-opioid agonist DADLE (20 nM). Co–incubation with the MEK inhibitors U0126 (500 nM) or PD 98059 (10 µM) blocked the increased ROS production seen with all three agonists. Direct activation of its receptor by EGF increased ROS production and PD 98059 blocked that increase, thus placing ERK downstream of the EGF receptor. Desferoxamine (DFO) which opens mitoKATP through direct activation of NOS also increased ROS. PD 98059 could not block DFO–induced ROS production, placing ERK upstream of NOS. In isolated hearts, ACh caused phosphorylation of both Akt and ERK. U0126 blocked phosphorylation of ERK but not of Akt. The PI3–K inhibitor wortmannin blocked both. Together these data indicate that ERK is located between Akt and NOS.