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Heidelberg, Australia

Katerelos M.,IBAS | Katerelos M.,University of Melbourne | Mudge S.J.,Austin Hospital | Stapleton D.,University of Melbourne | And 7 more authors.
Immunology and Cell Biology | Year: 2010

Activation of nuclear factor-kappa B (NF-B) is one of the most important pro-inflammatory mechanisms in disease. In this study, we show that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an intermediate in nucleoside metabolism, inhibits signalling by NF-B in three cell types, including bovine aortic endothelial cells (BAEC). The block in the NF-B signalling pathway occurred beyond degradation of IB-α and movement of p65 into the nucleus of BAEC. There was, however, reduced binding of NF-B from AICAR-treated cells to a B-consensus oligonucleotide, suggesting that part of the mechanism was a reduction in NF-B DNA-binding activity. Although AICAR is metabolized to ZMP and then adenosine, adenosine had no effect on activation of an NF-B reporter. ZMP, however, activates the metabolic stress-sensing AMP-activated protein kinase (AMPK). Transfection of active AMPK into BAEC reduced NF-B reporter activity compared with a kinase-dead mutant, suggesting that part of the ability of AICAR to inhibit NF-B signalling is due to activation of AMPK. Inhibition of NF-B signalling may be important in the anti-inflammatory action of drugs such as sulfasalazine and methotrexate, which led to the accumulation of AICAR within target cells. © 2010 Australasian Society for Immunology Inc. All rights reserved. Source


Alesutan I.,University of Tubingen | Munoz C.,University of Tubingen | Sopjani M.,University of Tubingen | Dermaku-Sopjani M.,University of Tubingen | And 6 more authors.
Biochemical and Biophysical Research Communications | Year: 2011

The inward rectifier K+ channel Kir2.1 participates in the maintenance of the cell membrane potential in a variety of cells including neurons and cardiac myocytes. Mutations of KCNJ2 encoding Kir2.1 underlie the Andersen-Tawil syndrome, a rare disorder clinically characterized by periodic paralysis, cardiac arrhythmia and skeletal abnormalities. The maintenance of the cardiac cell membrane potential is decreased in ischaemia, which is known to stimulate the AMP-activated serine/threonine protein kinase (AMPK). This energy-sensing kinase stimulates energy production and limits energy utilization. The present study explored whether AMPK regulates Kir2.1. To this end, cRNA encoding Kir2.1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKα1+AMPKβ1+AMPKγ1), of the constitutively active γR70QAMPK (α1β1γ1(R70Q)), of the kinase dead mutant αK45RAMPK (α1(K45R)β1γ1), or of the ubiquitin ligase Nedd4-2. Kir2.1 activity was determined in two-electrode voltage-clamp experiments. Moreover, Kir2.1 protein abundance in the cell membrane was determined by immunostaining and subsequent confocal imaging. As a result, wild type and constitutively active AMPK significantly reduced Kir2.1-mediated currents and Kir2.1 protein abundance in the cell membrane. Expression of wild type Nedd4-2 or of Nedd4-2S795A lacking an AMPK phosphorylation consensus sequence downregulated Kir2.1 currents. The effect of wild type Nedd4-2 but not of Nedd4-2S795A was significantly augmented by additional coexpression of AMPK. In conclusion, AMPK is a potent regulator of Kir2.1. AMPK is at least partially effective through phosphorylation of the ubiquitin ligase Nedd4-2. © 2011 Elsevier Inc. Source


Alesutan I.,University of Tubingen | Foller M.,University of Tubingen | Sopjani M.,University of Tubingen | DeRmaku-Sopjani M.,University of Tubingen | And 8 more authors.
Molecular Membrane Biology | Year: 2011

The heterotetrameric K+-channel KCNQ1/KCNE1 is expressed in heart, skeletal muscle, liver and several epithelia including the renal proximal tubule. In the heart, it contributes to the repolarization of cardiomyocytes. The repolarization is impaired in ischemia. Ischemia stimulates the AMP-activated protein kinase (AMPK), a serine/threonine kinase, sensing energy depletion and stimulating several cellular mechanisms to enhance energy production and to limit energy utilization. AMPK has previously been shown to downregulate the epithelial Na+ channel ENaC, an effect mediated by the ubiquitin ligase Nedd4-2. The present study explored whether AMPK regulates KCNQ1/KCNE1. To this end, cRNA encoding KCNQ1/KCNE1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKa1 + AMPKb1 + AMPKg1), of the constitutively active gR70QAMPK (a1b1g1(R70Q)), of the kinase dead mutant aK45RAMPK (a1(K45R)b1g1), or of the ubiquitin ligase Nedd4-2. KCNQ1/KCNE1 activity was determined in two electrode voltage clamp experiments. Moreover, KCNQ1 abundance in the cell membrane was determined by immunostaining and subsequent confocal imaging. As a result, wild type and constitutively active AMPK significantly reduced KCNQ1/KCNE1-mediated currents and reduced KCNQ1 abundance in the cell membrane. Similarly, Nedd4-2 decreased KCNQ1/KCNE1-mediated currents and KCNQ1 protein abundance in the cell membrane. Activation of AMPK in isolated perfused proximal renal tubules by AICAR (10 mM) was followed by significant depolarization. In conclusion, AMPK is a potent regulator of KCNQ1/KCNE1. © 2011 Informa UK, Ltd. Source

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