Reactive oxygen species (ROS) have been identified as central mediators in certain signalling events. In the heart, ROS have important functions in ischaemia/reperfusion-induced cardiac injury and in cytokine-stimulated hypertrophy. Extracellular signal-regulated kinase (ERK) is one of the ROS-responsive serine/threonine kinases. Previous studies showed that tyrosine kinases and small G proteins are involved in the activation of ERK by ROS; however, the initial target protein of ROS that leads to ERK activation remains unknown. Here we show that inhibition of the betagamma-subunit of G protein (G betagamma) attenuates hydrogen peroxide (H2O2)-induced ERK activation in rat neonatal cardiomyocytes. The G betagamma-responsive ERK activation induced by H2O2 is independent of ligands binding to Gi-coupled receptors, but requires phosphatidylinositol-3-kinase and Src activation. In in vitro studies, however, treatment with H2O2 increases [35S]GTP-gammaS binding to cardiac membranes and directly activates purified heterotrimeric Gi and Go but not Gs. Analysis using heterotrimeric Go and its individual subunits indicates that H2O2 modifies G alpha(o) but not G betagamma, which leads to subunit dissociation. We conclude that G alpha(i) and G alpha(o) are critical targets of oxidative stress for activation of ERK.

In the present study, we examined signal transduction mechanism of reactive oxygen species (ROS) production and the role of ROS in angiotensin II-induced activation of mitogen-activated protein kinases (MAPKs) in rat neonatal cardiomyocytes. Among three MAPKs, c-Jun NH 2 -terminal kinase (JNK) and p38 MAPK required ROS production for activation, as an NADPH oxidase inhibitor, diphenyleneiodonium, inhibited the activation.

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Abstract-In neonatal cardiomyocytes, activation of the G q -coupled ␣ 1 -adrenergic receptor (␣ 1 AR) induces hypertrophy by activating mitogen-activated protein kinases, including c-Jun NH 2 -terminal kinase (JNK). Here, we show that JNK activation is essential for ␣ 1 AR-induced hypertrophy, in that ␣ 1 AR-induced hypertrophic responses, such as reorganization of the actin cytoskeleton and increased protein synthesis, could be blocked by expressing the JNK-binding domain of JNK-interacting protein-1, a specific inhibitor of JNK. We also identified the classes and subunits of G proteins that mediate ␣ 1 AR-induced JNK activation and hypertrophic responses by generating several recombinant adenoviruses that express polypeptides capable of inhibiting the function of specific G-protein subunits. ␣ 1 AR-induced JNK activation was inhibited by the expression of carboxyl terminal regions of G␣ q , G␣ 12 , and G␣ 13 . JNK activation was also inhibited by the G␣ q/11 -or G␣ 12/13 -specific regulator of G-protein signaling (RGS) domains and by C3 toxin but was not affected by treatment with pertussis toxin or by expression of the carboxyl terminal region of G protein-coupled receptor kinase 2, a polypeptide that sequesters G␤␥. ␣ 1 AR-induced hypertrophic responses were inhibited by G␣ q/11 -and G␣ 12/13 -specific RGS domains, C3 toxin, and the carboxyl terminal region of G protein-coupled receptor kinase 2 but not by pertussis toxin. Activation of Rho was inhibited by carboxyl terminal regions of G␣ 12 and G␣ 13 but not by G␣ q . Our findings suggest that ␣ 1 AR-induced hypertrophic responses are mediated in part by a G␣ 12/13 -Rho-JNK pathway, in part by a G q/11 -JNK pathway that is Rho independent, and in part by a G␤␥ pathway that is JNK independent. Key Words: c-Jun NH 2 -terminal kinase Ⅲ G 12 family G proteins Ⅲ ␣ 1 -adrenergic receptors Ⅲ hypertrophy I n cardiomyocytes, stimulation of G-protein-coupled receptors activates mitogen-activated protein kinases, including c-Jun NH 2 -terminal kinase (JNK), which can subsequently trigger hypertrophic responses. 1 ␣ 1 -Adrenergic receptors (␣ 1 ARs) are G q -coupled receptors expressed in the heart, and they are thought to play a role in cardiac hypertrophy. Significant evidence points to an important role for G␣ q in receptor-induced hypertrophic responses. For example, ␣ 1 AR-induced hypertrophy can be inhibited by neutralizing antibodies that recognize G␣ q . In addition, a mutated M1 muscarinic acetylcholine receptor with impaired coupling to G q , in contrast to the wild-type receptor, is unable to induce hypertrophy. 2,3 It has also been reported that cardiac hypertrophy can be induced by expressing a constitutively active mutant of G␣ q . 4 In total, these results suggest that G␣ q plays an essential role in G q -coupled receptor-mediated hypertrophy, a conclusion strengthened by evidence that overexpression of protein kinase C␤, which is activated by diacylglycerol, can induce hypertrophy in a transgenic animal model. 5 Recent reports have demonstrated that var...