Mitochondrial ATP production is necessary for activation of the extracellular-signal-regulated kinases during ischaemia/reperfusion in rat myocyte-derived H9c2 cells

To search for the stimuli involved in activating the mitogen-activated prot ein kinases (MAPKs) during ischaemia and reperfusion, we simulated the even t in a system in vitro conducive to continuous and non-invasive measurement s of several major perturbations that occur at the time: O-2 tension, mitoc hondrial respiration and energy status. Using H9c2 cells (a clonal line der ived from rat heart), we found that activation of the extracellular signal- regulated MAPKs (ERKs) on reoxygenation was abolished if the mitochondria w ere inhibited prior to and during reoxygenation. Re-introduction of O-2 pcv sc is therefore not sufficient to activate the ERKs. Recovery and maintena nce of cellular ATP levels by mitochondrial respiration is necessary, altho ugh ATP recovery alone is not sufficient. ERK activation by H2O2, but not p horbol esters, was also sensitive to mitochondrial inhibition. Thus, reoxyg enation and H2O2-mediated oxidative stress share a mechanism of ERK activat ion that is ATP- or mitochondrion-dependent, and this common feature sugges ts that the reoxygenation response is mediated by reactive oxygen species. A correlation between ERK activity and ATP levels was also found during the anoxic phase of ischaemia, an effect that was not due to substrate limitat ion for the kinases. Our results reveal the importance of cellular metaboli sm in ERK activation, and introduce ATP as a novel participant in the mecha nisms underlying the ERK cascade.