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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Reactive oxygen species are proposed to work as intracellular mediators. One of their target proteins is the ␣ subunit of heterotrimeric GTP-binding proteins ( (3, 4). The increase in H 2 O 2 by platelet-derived growth factor receptor stimulation requires the activation of phosphatidylinositol 3-kinase (PI3K) and is possibly mediated by PI3K/Rac/NADPH oxidase pathway (3). The increase in H 2 O 2 was also observed by stimulation of G protein-coupled receptors such as angiotensin II (5), lysophosphatidic acid (6), and thrombin receptors (7). The generated H 2 O 2 is found to inactivate protein-tyrosine phosphatase 1B (PTP-1B) by modifying the cysteine residue located at catalytic moiety (8). However, the analysis of PTP-1B from H 2 O 2 -treated cells revealed that the cysteine residue of PTP-1B is not sulfenic acid-but glutathione-conjugated cysteine (9). Therefore, the cysteine residue of PTP-1B is at first modified by H 2 O 2 and then reacts with glutathione. This modification of cysteine leads to the inactivation of phosphatase activity of PTP-1B. Thus, the increase in H 2 O 2 indirectly changes the phosphorylation-dephosphorylation state on the tyrosine residue of proteins implicated in signal transduction (10).ROS can also be generated upon pathophysiological conditions. For instance, ROS are generated in a large amount on ischemia/reperfusion and can influence many intracellular signaling processes (11). Because the inclusion of superoxide dismutase mimics or antioxidant attenuates the cellular injury caused by ischemia/reperfusion, the prevention and mechanism of ROS generation is critical for the understanding of ischemia/reperfusion injury. The treatment with H 2 O 2 is frequently used for mimicking oxidative stress such as ischemia/ reperfusion, since H 2 O 2 freely enters cells like H 2 O (1, 2). The treatment with H 2 O 2 activates mitogen-activated protein kinase (MAPKs) such as ERK, c-Jun NH 2 -terminal kinase, and p38 MAPK and promotes or inhibits cellular injury (12).We have previously reported that the treatment of rat neonatal myocytes with H 2 O 2 activates G i and G o in a receptorindependent manner, leading to the increased activity of ERK (13). However, the molecular mechanism of G i and G o activation by H 2 O 2 is still not clear, especially about the modified amino acids. We demonstrate in the present study that H 2 O 2 is converted to more reactive species in the presence of Fe 2ϩ and modifies specific cysteine residues that exist only in G␣ i and G␣ o . The modification of two cysteine residues results in subunit dissociation of G i and increase in GTP␥S binding. . generator (KO 2 ) and PMSF were purchased from Sigma, and a singlet oxygen generator EXPERIMENTAL PROCEDURES Materials-[

In the present study, we examined the roles of G 12 , G 13 , G q , and G i in endothelin-1-induced hypertrophic responses. Endothelin-1 stimulation activated extracellular signal-regulated kinase (ERK) and c-Jun NH 2 -terminal kinase (JNK) in cultured rat neonatal myocytes. The activation of JNK, but not ERK, was inhibited by the expression of carboxyl terminal regions of G␣ 12 and G␣ 13 . JNK activation was also inhibited by expression of the G␣ 12 /G␣ 13 -specific inhibitor regulator of G protein signaling (RGS) domain of p115RhoGEF and the G␣ q -specific inhibitor RGS domain of the G protein-coupled receptor kinase 2 (GRK2-RGS). JNK activation was not, however, inhibited by expression of the carboxyl terminal region of G protein-coupled receptor kinase 2 (GRK2-ct), which is a G␤␥-sequestering polypeptide. Additionally, JNK activation but not ERK activation was inhibited by the expression of C3 exoenzyme that inactivates small GTPase Rho. These results suggest that JNK activation by G␣ 12 , G␣ 13 , and G␣ q is involved in Rho. On the other hand, ERK activation was inhibited by pertussis toxin treatment, the receptor-G i uncoupler, and GRK2-ct. Thus, ERK was activated by G␣ i -and G␤␥-dependent pathways. These results clearly demonstrate that differential pathways activate JNK and ERK.

SummaryIn Drosophila, the melanization reaction is an important defense mechanism against injury and invasion of microorganisms. Drosophila tyrosine hydroxylase (TH, also known as Pale) and dopa decarboxylase (Ddc), key enzymes in the dopamine synthesis pathway, underlie the melanin synthesis by providing the melanin precursors dopa and dopamine, respectively. It has been shown that expression of Drosophila TH and Ddc is induced in various physiological and pathological conditions, including bacterial challenge; however, the mechanism involved has not been fully elucidated. Here, we show that ectopic activation of p38 MAPK induces TH and Ddc expression, leading to upregulation of melanization in the Drosophila cuticle. This p38-dependent melanization was attenuated by knockdown of TH and Ddc, as well as by that of Drosophila HR38, a member of the NR4A family of nuclear receptors. In mammalian cells, p38 phosphorylated mammalian NR4As and Drosophila HR38 and potentiated these NR4As to transactivate a promoter containing NR4A-binding elements, with this transactivation being, at least in part, dependent on the phosphorylation. This suggests an evolutionarily conserved role for p38 MAPKs in the regulation of NR4As. Thus, p38-regulated gene induction through NR4As appears to function in the dopamine synthesis pathway and may be involved in immune and stress responses.

The discovery and optimization of Δ-5 desaturase (D5D) inhibitors are described. Investigation of the 1,3-oxazolidin-2-one scaffold was inspired by a pharmacophore model constructed from the common features of several hit compounds, resulting in the identification of 3,5-diphenyl-1,3-oxazolidin-2-one 5h as a novel lead showing potent in vitro activity. Subsequent optimization focused on the modification of two metabolic sites, which provided (4S,5S)-5i, a derivative with improved metabolic stability. Moreover, adding a substituent into the upper phenyl moiety further enhanced the intrinsic activity, which led to the discovery of 5-[(4S,5S)-5-(4fluorophenyl)-4-methyl-2-oxo-1,3-oxazolidin-3-yl]benzene-1,3-dicarbonitrile (4S,5S)-5n, endowed with excellent D5D binding affinity, cellular activity, and high oral bioavailability in a mouse. It exhibited robust in vivo hepatic arachidonic acid/dihomo-γ-linolenic acid ratio reduction (a target engagement marker) in an atherosclerosis mouse model. Finally, an asymmetric synthetic procedure for this compound was established.

Obesity is now recognized as a state of chronic low-grade inflammation and is called as metabolic inflammation. Delta-5 desaturase (D5D) is an enzyme that metabolizes dihomo-γ-linolenic acid (DGLA) to arachidonic acid (AA). Thus, D5D inhibition increases DGLA (precursor to anti-inflammatory eicosanoids) while decreasing AA (precursor to pro-inflammatory eicosanoids), and could result in synergistic improvement in the low-grade inflammatory state. Here, we demonstrate reduced insulin resistance and the anti-obesity effect of a D5D selective inhibitor (compound-326), an orally active small-molecule, in a high-fat diet-induced obese (DIO) mouse model. In vivo D5D inhibition was confirmed by determining changes in blood AA/DGLA profiles. In DIO mice, chronic treatment with compound-326 lowered insulin resistance and caused body weight loss without significant impact on cumulative calorie intake. Decreased macrophage infiltration into adipose tissue was expected from mRNA analysis. Increased daily energy expenditure was also observed following administration of compound-326, in line with sustained body weight loss. These data indicate that the novel D5D selective inhibitor, compound-326, will be a new class of drug for the treatment of obese and diabetic patients.

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