endothelin-1 and phorbolol-myristate-acetate

The increase in endothelin-1 (ET-1) and the decrease in endothelial nitric oxide synthase (eNOS) both induce vasoconstriction and lead to molecular changes associated with diabetes mellitus and atherosclerosis. Glucagon-like peptide-1 (GLP-1) activation stimulates insulin secretion and may prevent atherosclerosis by increasing eNOS synthesis. However, there is paucity of information on the effect of GLP-1 activation on ET-1 expression. This study was conducted to address this issue.. Human umbilical vein endothelial cells (HUVECs) were incubated with different concentrations of liraglutide, a GLP-1 agonist, and the expression of ET-1 and eNOS and activity of NF-κB were measured. Liraglutide, in a concentration-dependent manner, was observed to promote eNOS expression and to inhibit ET-1 expression both at mRNA and protein levels. Liraglutide also inhibited NF-κB phosphorylation and its translocation from cytoplasm to the nucleus. To ascertain the role of NF-κB activation in the altered expression of ET-1 and eNOS, we treated HUVECs with phorbol 12-myristate 13-acetate (PMA). PMA activated NF-κB and reversed the effects of liraglutide on eNOS and ET-1 expression. The effects of PMA on eNOS and ET-1 expression were reproduced in experiments wherein cells were treated with TNF-α. Further, we measured the generation of IL-6, apowerful pro-inflammatory molecule released by endothelial cells, as a measure of cellular function. PMA increased IL-6 generation, and this effect was blocked by liraglutide.. Our observations suggest liraglutide suppresses ET-1 expression by inhibiting the phosphorylation of NF-κB. This mechanism may underlie the potential anti-atherosclerotic effects of GLP-1 agonists. Of note, these effects of liraglutide were seen in an in vitro setting wherein cellular glucose concentrations were elevated.

Topics: Cells, Cultured; Endothelin-1; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Human Umbilical Vein Endothelial Cells; Humans; Interleukin-6; Liraglutide; NF-kappa B; Nitric Oxide Synthase Type III; Receptors, Glucagon; Tetradecanoylphorbol Acetate

ERK1 and ERK2 (ERK1/2) are central to the regulation of cell division, growth and survival. They are activated by phosphorylation of the Thr- and the Tyr- residues in their Thr-Glu-Tyr activation loops. The dogma is that dually-phosphorylated ERK1/2 constitute the principal activities in intact cells. We previously showed that, in neonatal rat cardiac myocytes, endothelin-1 and phorbol 12-myristate 13-acetate (PMA) powerfully and rapidly (maximal at ~5 min) activate ERK1/2. Here, we show that dually-phosphorylated ERK1/2 rapidly (< 2 min) appear in the nucleus following stimulation with endothelin-1. We characterized the active ERK1/2 species in myocytes exposed to endothelin-1 or PMA using MonoQ FPLC. Unexpectedly, two peaks of ERK1 and two peaks of ERK2 activity were resolved using in vitro kinase assays. One of each of these represented the dually-phosphorylated species. The other two represented activities for ERK1 or ERK2 which were phosphorylated solely on the Thr- residue. Monophosphothreonyl ERK1/2 represented maximally ~30% of total ERK1/2 activity after stimulation with endothelin-1 or PMA, and their k(cat) values were estimated to be minimally ~30% of the dually-phosphorylated species. Appearance of monophosphothreonyl ERK1/2 was rapid but delayed in comparison with dually-phosphorylated ERK1/2. Of 10 agonists studied, endothelin-1 and PMA were most effective in terms of ERK1/2 activation and in stimulating the appearance of monophosphothreonyl and dually-phosphorylated ERK1/2. Thus, enzymically active monophosphothreonyl ERK1/2 are formed endogenously following activation of the ERK1/2 cascade and we suggest that monophosphothreonyl ERK1/2 arise by protein tyrosine phosphatase-mediated dephosphorylation of dually-phosphorylated ERK1/2.

Topics: Animals; Cells, Cultured; Endothelin-1; Enzyme Activation; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myocytes, Cardiac; Phosphorylation; Rats; Rats, Sprague-Dawley; Signal Transduction; Tetradecanoylphorbol Acetate

RGS5 is a member of regulators of G protein signaling (RGS) proteins that attenuate heterotrimeric G protein signaling by functioning as GTPase-activating proteins (GAPs). We investigated phosphorylation of RGS5 and the resulting change of its function. In 293T cells, transiently expressed RGS5 was phosphorylated by endogenous protein kinases in the basal state. The phosphorylation was enhanced by phorbol 12-myristate 13-acetate (PMA) and endothelin-1 (ET-1), and suppressed by protein kinase C (PKC) inhibitors, H7, calphostin C and staurosporine. These results suggest involvement of PKC in phosphorylation of RGS5. In in vitro experiments, PKC phosphorylated recombinant RGS5 protein at serine residues. RGS5 protein phosphorylated by PKC showed much lower binding capacity for and GAP activity toward Galpha subunits than did the unphosphorylated RGS5. In cells expressing RGS5, the inhibitory effect of RGS5 on ET-1-induced Ca(2+) responses was enhanced by staurosporine. Mass spectrometric analysis of the phosphorylated RGS5 revealed that Ser166 was one of the predominant phosphorylation sites. Substitution of Ser166 by aspartic acid abolished the binding capacity to Galpha subunits and the GAP activity, and markedly reduced the inhibitory effect on ET-1-induced Ca(2+) responses. These results indicate that phosphorylation at Ser166 of RGS5 by PKC causes loss of the function of RGS5 in G protein signaling. Since this serine residue is conserved in RGS domains of many RGS proteins, the phosphorylation at Ser166 by PKC might act as a molecular switch and have functional significance.

Topics: Calcium; Cell Line; Electrophoresis, Polyacrylamide Gel; Endothelin-1; GTP-Binding Protein alpha Subunits; Humans; Peptide Fragments; Phosphorylation; Protein Binding; Protein Kinase C; Protein Kinase Inhibitors; Recombinant Proteins; RGS Proteins; Serine; Signal Transduction; Tetradecanoylphorbol Acetate

Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors that mediate phospholipase D (PLD) activation in brain, but the mechanism underlying this response remains unclear. Here we used primary cultures of astrocytes as a cell model to explore the mechanism that links mGluRs to PLD. Glutamate activated both phospholipase C (PLC) and PLD with equal potency and this effect was mimicked by L-cysteinesulfinic acid, a putative neurotransmitter previously shown to activate mGluRs coupled to PLD, but not PLC, in adult brain. PLD activation by glutamate was dependent on Ca(2+) mobilization and fully blocked by both protein kinase C (PKC) inhibitors and PKC down-regulation, suggesting that PLD activation is secondary to PLC stimulation. Furthermore, brefeldin A, an inhibitor of ADP-ribosylation factor (ARF) activation, partially inhibited the activation of PLD by glutamate. By contrast, pretreatment of astrocytes with Clostridium difficile toxin B, which inactivates small G proteins of the Rho family (Rho, Rac, and Cdc42), had no effect on PLD stimulation by glutamate. Taken together, these results indicate that PLD activation by mGluRs in astrocytes is dependent on PKC and small G proteins of the ARF family, but does not require Rho proteins.

Topics: ADP-Ribosylation Factors; Animals; Astrocytes; Bacterial Proteins; Bacterial Toxins; Brefeldin A; Cells, Cultured; Chelating Agents; Cysteine; DNA; Dose-Response Relationship, Drug; Egtazic Acid; Endothelin-1; Glutamic Acid; Indoles; Maleimides; Phospholipase D; Protein Kinase C; Protein Synthesis Inhibitors; Rats; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; rho GTP-Binding Proteins; Stress Fibers; Sulfenic Acids; Tetradecanoylphorbol Acetate; Type C Phospholipases