Biguanides such as metformin have previously been shown to antagonize hepatic glucagon-stimulated cyclic AMP (cAMP) signalling independently of AMP-activated protein kinase (AMPK) via direct inhibition of adenylate cyclase by AMP. Here we show that incubation of hepatocytes with the small-molecule AMPK activator 991 decreases glucagon-stimulated cAMP accumulation, cAMP-dependent protein kinase (PKA) activity and downstream PKA target phosphorylation. Moreover, incubation of hepatocytes with 991 increases the Vmax of cyclic nucleotide phosphodiesterase 4B (PDE4B) without affecting intracellular adenine nucleotide concentrations. The effects of 991 to decrease glucagon-stimulated cAMP concentrations and activate PDE4B are lost in hepatocytes deleted for both catalytic subunits of AMPK. PDE4B is phosphorylated by AMPK at three sites, and by site-directed mutagenesis, Ser304 phosphorylation is important for activation. In conclusion, we provide a new mechanism by which AMPK antagonizes hepatic glucagon signalling via phosphorylation-induced PDE4B activation.

1 The possible mechanisms of action of the inhibitory effect of gomisin C on the respiratory burst of rat neutrophils in vitro was investigated. 2 The peptide formyl-Met-Leu-Phe (FMLP) induced superoxide anion (02 -) formation and 02 consumption, which was inhibited by gomisin C in a concentration-dependent manner (IC50 21.5 ± 4.2 fg ml-' for 02-formation). Gomisin C also suppressed 02-formation and 02 consumption at low concentrations of phorbol myristate acetate (PMA) with an IC50 value of 26.9 ± 2.1 Pg ml-' for°2-formation. However, gomisin C did not affect the responses induced by a high concentration of PMA. 3 Gomisin C had no effect on°2-generation and uric acid formation in the xanthine-xanthine oxidase system, and failed to alter 02-generation during dihydroxyfumaric acid (DHF) autoxidation, indicating that it does not scavenge superoxide. 4 Like trifluoperazine (TFP), gomisin C attenuated the activity of PMA-activated neutrophil particulate NADPH oxidase in a concentration-dependent manner.5 Gomisin C reduced the elevations of cytosolic free Ca2+ in neutrophils stimulated by FMLP in the presence or absence of EDTA. Cyclopiazonic acid (CPA) induced the release of Ca2+ from intracellular stores and this was also reduced by gomisin C. However, the Ca2+ influx pathway activated by CPA was not affected by gomisin C. 6 The cellular cyclic AMP level was markedly increased by forskolin, but not by gomisin C. Moreover, the inositol phosphate levels in FMLP-activated neutrophils were not affected by gomisin C. 7 These results show that the inhibitory action of gomisin C on the respiratory burst is not mediated by changes in cellular cyclic AMP or in inositol phosphates, or by scavenging°2 released from neutrophils, but may be mediated partly by the suppression of NADPH oxidase and partly by the decrease of cytosolic Ca2+ released from an agonist-sensitive intracellular store.

1 The possible mechanisms of the inhibitory e ect of ethyl 2-(3-hydroxyanilino)-4-oxo-4,5-dihydrofuran-3-carboxylate (HAJ11) on the respiratory burst of rat neutrophils in vitro was investigated. 2 HAJ11 caused a reversible and a concentration-dependent inhibition of formyl-Met-Leu-Phe (fMLP)-induced superoxide anion (O 2 ⋅7 ) generation (IC 50 4.9+0.7 mM) and O 2 consumption (IC 50 4.9+1.5 mM). Concanavalin A (Con A)-and NaF-induced O 2 ⋅7 generation were also suppressed by HAJ11. However, HAJ11 was a weak inhibitor of the phorbol 12-myristate 13-acetate (PMA)-induced responses. 3 HAJ11 did not scavenge the O 2 ⋅7 generation in the xanthine-xanthine oxidase system and dihydroxyfumaric acid (DHF) autoxidation. 4 HAJ11 showed no activity on fMLP-induced inositol phosphates formation and [Ca 2+ ] i elevation in intact neutrophils. In addition, HAJ11 had no e ect on neutrophil cytosolic phospholipase C (PLC) activity. 5 HAJ11 reduced fMLP-induced phosphatidic acid (PA) (IC 50 29.1+6.5 mM) and phosphatidylethanol (PEt) (IC 50 22.6+1.9 mM) formation in a concentration-dependent manner. HAJ11 also reduced protein tyrosine phosphorylation in neutrophils stimulated by fMLP. 6 HAJ11 was a weak inhibitor of neutrophil cytosolic protein kinase C (PKC) activity, and had a negligible e ect on brain PKC. Cellular cyclic nucleotides levels were not altered by HAJ11. In addition, HAJ11 did not a ect protein kinase A (PKA) activity. 7 HAJ11 had no e ect on the O 2 ⋅7 generation of PMA-activated and arachidonic acid (AA)-activated NADPH oxidase preparations. 8 Taken together these results indicate that the inhibition of respiratory burst by HAJ11 probably mainly occurs through inhibition of protein tyrosine phosphorylation and phospholipase D (PLD) activity.

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