okadaic-acid and rottlerin

Endothelial nitric-oxide synthase (eNOS) utilizes l-arginine as its principal substrate, converting it to l-citrulline and nitric oxide (NO). l-Citrulline is recycled to l-arginine by two enzymes, argininosuccinate synthase (AS) and argininosuccinate lyase, providing the substrate arginine for eNOS and NO production in endothelial cells. Together, these three enzymes, eNOS, AS, and argininosuccinate lyase, make up the citrulline-NO cycle. Although AS catalyzes the rate-limiting step in NO production, little is known about the regulation of AS in endothelial cells beyond the level of transcription. In this study, we showed that AS Ser-328 phosphorylation was coordinately regulated with eNOS Ser-1179 phosphorylation when bovine aortic endothelial cells were stimulated by either a calcium ionophore or thapsigargin to produce NO. Furthermore, using in vitro kinase assay, kinase inhibition studies, as well as protein kinase Cα (PKCα) knockdown experiments, we demonstrate that the calcium-dependent phosphorylation of AS Ser-328 is mediated by PKCα. Collectively, these findings suggest that phosphorylation of AS at Ser-328 is regulated in accordance with the calcium-dependent regulation of eNOS under conditions that promote NO production and are in keeping with the rate-limiting role of AS in the citrulline-NO cycle of vascular endothelial cells.

Topics: Acetophenones; Amino Acid Substitution; Animals; Aorta; Argininosuccinate Synthase; Benzopyrans; Bradykinin; Calcium; Calcium Signaling; Cattle; Cells, Cultured; Endothelial Cells; Enzyme Activation; Gene Knockdown Techniques; Indoles; Isoenzymes; Maleimides; Mutagenesis, Site-Directed; Nitric Oxide; Nitric Oxide Synthase Type III; Okadaic Acid; Phosphorylation; Protein Kinase C-alpha; Protein Phosphatase 1; Protein Phosphatase 2; Protein Processing, Post-Translational; RNA Interference; Serine

Human neutrophils differ from other cells by containing high amount of IkappaBalpha in the nucleus, and this increased nuclear IkappaBalpha accumulation is associated with the inhibition of NFkappaB activity and increased apoptosis. However, the mechanisms regulating NFkappaB activation and IkappaBalpha degradation in human neutrophils are little understood. The objective of this study was to provide a further insight into the mechanisms regulating NFkappaB activity and IkappaBalpha degradation in human neutrophils. We show that okadaic acid (OA), an inhibitor of protein phosphatases PP1 and PP2A, induces sustained activation of NFkappaB and degradation of the nuclear IkappaBalpha, and increases interleukin-8 expression in the neutrophils. Furthermore, inhibitors of protein kinase C-delta (PKCdelta) and IkappaB kinase (IKK) inhibit the OA-induced activation of NFkappaB. Collectively, our results indicate that in human neutrophils, the sustained activation of NFkappaB is regulated by a continuous phosphorylation and degradation of the nuclear IkappaBalpha.

Topics: Acetophenones; Benzopyrans; Cell Nucleus; Cells, Cultured; Dose-Response Relationship, Drug; Humans; I-kappa B Proteins; Interleukin-8; Lipopolysaccharides; Mitogen-Activated Protein Kinases; Neutrophil Activation; Neutrophils; NF-kappa B; NF-KappaB Inhibitor alpha; Okadaic Acid; p38 Mitogen-Activated Protein Kinases; Phosphoprotein Phosphatases; Protein Kinase C; Sulindac; Transcription, Genetic; Tumor Necrosis Factor-alpha