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Spatiotemporal regulation of cAMP within the cell is required to achieve receptor-specific responses. The mechanism through which the cell selects a specific response to newly synthesized cAMP is not fully understood. In hepatocyte plasma membranes, we identified two functional and independent cAMP-responsive signaling protein macrocomplexes that produce, use, degrade, and regulate their own nondiffusible (sequestered) cAMP pool to achieve their specific responses. Each complex responds to the stimulation of an adenosine G protein-coupled receptor (Ado-GPCR), bound to either A2A or A2B , but not simultaneously to both. Each isoprotein involved in each signaling cascade was identified by measuring changes in cAMP levels after receptor activation, and its participation was confirmed by antibody-mediated inactivation. A2A -Ado-GPCR selective stimulation activates adenylyl cyclase 6 (AC6), which is bound to AKAP79/150, to synthesize cAMP which is used by two other AKAP79/150-tethered proteins: protein kinase A (PKA) and phosphodiesterase 3A (PDE3A). In contrast, A2B -Ado-GPCR stimulation activates D-AKAP2-attached AC5 to generate cAMP, which is channeled to two other D-AKAP2-tethered proteins: guanine-nucleotide exchange factor 2 (Epac2) and PDE3B. In both cases, prior activation of PKA or Epac2 with selective cAMP analogs prevents de novo cAMP synthesis. In addition, we show that cAMP does not diffuse between these protein macrocomplexes or 'signalosomes'. Evidence of coimmunoprecipitation and colocalization of some proteins belonging to each signalosome is presented. Each signalosome constitutes a minimal functional signaling unit with its own machinery to synthesize and regulate a sequestered cAMP pool. Thus, each signalosome is devoted to ensure the transmission of a unique and unequivocal message through the cell.

Renin-angiotensin system (RAS) activation promotes oxidative stress which increases the risk of cardiac dysfunction in metabolic syndrome (MetS) and favors local insulin resistance. Fibrates regulate RAS improving MetS, type-2 diabetes and cardiovascular diseases. We studied the effect of fenofibrate treatment on the myocardic signaling pathway of Angiotensin II (Ang II)/Angiotensin II type 1 receptor (AT1) and its relationship with oxidative stress and myocardial insulin resistance in MetS rats under heart ischemia. Control and MetS rats were assigned to the following groups: (a) sham; (b) vehicle-treated myocardial infarction (MI) (MI-V); and (c) fenofibrate-treated myocardial infarction (MI-F). Treatment with fenofibrate significantly reduced triglycerides, non-high density lipoprotein cholesterol (non-HDL-C), insulin levels and insulin resistance index (HOMA-IR) in MetS animals. MetS and MI increased Ang II concentration and AT1 expression, favored myocardial oxidative stress (high levels of malondialdehyde, overexpression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4), decreased total antioxidant capacity and diminished expression of superoxide dismutase (SOD)1, SOD2 and catalase) and inhibited expression of the insulin signaling cascade: phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PkB, also known as Akt)/Glut-4/endothelial nitric oxide synthase (eNOS). In conclusion, fenofibrate treatment favors an antioxidant environment as a consequence of a reduction of the Ang II/AT1/NOX4 signaling pathway, reestablishing the cardiac insulin signaling pathway. This might optimize cardiac metabolism and improve the vasodilator function during myocardial ischemia.

Acute ethanol intoxication increased triacylglycerides (TAG) and thiobarbituric acid reactive substances (TBARS) in liver and promoted the liberation of epinephrine. Four non-steroidal anti-inflammatory drugs (NSAIDs) - aspirin, naproxen, nimesulide and piroxicam - prevented this increase in TAG and TBARS. Because fatty acids provided by adipose tissue contribute substantially to elevated hepatic TAG in ethanol-intoxicated rats, it was thought that the NSAIDs might reduce epinephrine-stimulated lipolysis in these rats. Isolated rat adipocytes were activated with epinephrine in the presence or absence of the NSAIDs. The NSAIDs inhibited epinephrine-stimulated lipolysis. These drugs did not modify the binding of dihydroalprenolol (β-adrenergic agonist) to their receptors in isolated guinea-pig liver membranes. The NSAIDs, at concentrations 3000-fold lower than that of cAMP, inhibited stimulated lipolysis by this messenger. In conclusion, aspirin, naproxen, nimesulide and piroxicam reduce the release of fatty acids from adipose tissue to the liver by inhibiting the epinephrine-stimulated lipolysis, and this, in part, explains the protective action of these NSAIDs against hepatic signs of acute ethanol intoxication.

Hepatic surgery requires, under diverse circumstances, periods of ischemia and reperfusion (I-R) such as those present in liver resection, hepatic injury, and liver transplantation. The objective of the present work was to conduct an experimental study to evaluate the effect of hepatic preconditioning (HPC) on modulation of the I-R injury.Male Wistar rats were distributed into the following three study groups: group 1, simulated or sham; group 2, submitted to a 30-min period of total warm ischemia and a reperfusion phase, and group 3, in which we carried out 10-min preconditioning of warm ischemia and 10 min of reperfusion prior to the total ischemia period for a total of 60 min and the reperfusion phase. We obtained liver biopsies for thiobarbituric acid reactive substances (TBARS) (MDA, thiobarbituric acid adducts) and for blood sample determinations in serum of liver-cell enzymes such as alanine aminotransferase and aspartate aminotransferase during a 24-h time course.We observed a decrease in the variables studied in group 3 (HPC) as well as of serum liver enzymes and TBARS levels such as indirect oxidative stress indicators upon comparison with group 2 animals submitted to total hepatic I-R.HPC is an efficient surgical strategy for decreasing the elevation of hepatic enzymes and indirect lipoperoxidation indicators in an I-R model. Controlled clinical studies should be performed to determine its functional properties and clinical applicability.

Among many actions assigned to taurine (Tau), the most abundant amino acid in numerous mammalian tissues, it prevents high-fat diet-induced obesity with increasing resting energy expenditure. To sustain this Tau action, the goal of the present study was to explore the acute effects of Tau on baseline and on adrenaline, insulin and their second messengers to modulate lipolysis in white adipose tissue (WAT) cells from rat epididymis. The Tau effects in this topic were compared with those recorded with Gly, Cys and Met. Tau on its own did not modify baseline lipolysis. Tau raised isoproterenol- and dibutyryl-cAMP (Bt2cAMP)-activated glycerol release. Gly diminished Bt2cAMP-activated glycerol release, and Cys and Met had no effect. Cyclic AMP-dependent activation of protein kinase A (PKA) in cell-free extracts decreased slightly by Gly and was unaltered by Cys, Met, and Tau. PKA catalytic activity in cell-free extracts was stimulated by Tau and unchanged by Cys, Gly and Met. Gly and Tau effects on PKA disappeared when these amino acids were withdrawn by gel filtration. Insulin-mediated NADPH-oxidase (NOX) raises H2O2 pool, which promotes PKA subunit oxidation, and precludes its cAMP activation; thus, lipolysis is diminished. Tau, but not Cys, Gly and Met, inhibited, by as much as 70%, insulin-mediated H2O2 pool increase. These data suggested that Tau raised lipolysis in adipocytes by two mechanisms: stimulating cAMP-dependent PKA catalytic activity and favoring PKA activation by cAMP as a consequence of lowering the H2O2 pool.

Catecholamines in adipose tissue promote lipolysis via cAMP, whereas insulin stimulates lipogenesis. Here we show that H(2)O(2) generated by insulin in rat adipocytes impaired cAMP-mediated amplification cascade of lipolysis. These micromolar concentrations of H(2)O(2) added before cAMP suppressed cAMP activation of type IIbeta cyclic AMP-dependent protein kinase (PKA) holoenzyme, prevented hormone-sensitive lipase translocation from cytosol to storage droplets, and inhibited lipolysis. Similarly, H(2)O(2) impaired activation of type IIalpha PKA holoenzyme from bovine heart and from that reconstituted with regulatory IIalpha and catalytic alpha subunits. H(2)O(2) was ineffective (a) if these PKA holoenzymes were preincubated with cAMP, (b) if added to the catalytic alpha subunit, which is active independently of cAMP activation, and (c) if the catalytic alpha subunit was substituted by its C199A mutant in the reconstituted holoenzyme. H(2)O(2) inhibition of PKA activation remained after H(2)O(2) elimination by gel filtration but was reverted with dithiothreitol or with thioredoxin reductase plus thioredoxin. Electrophoresis of holoenzyme in SDS gels showed separation of catalytic and regulatory subunits after cAMP incubation but a single band after H(2)O(2) incubation. These data strongly suggest that H(2)O(2) promotes the formation of an intersubunit disulfide bond, impairing cAMP-dependent PKA activation. Phylogenetic analysis showed that Cys-97 is conserved only in type II regulatory subunits and not in type I regulatory subunits; hence, the redox regulation mechanism described is restricted to type II PKA-expressing tissues. In conclusion, phylogenetic analysis results, selective chemical behavior, and the privileged position in holoenzyme lead us to suggest that Cys-97 in regulatory IIalpha or IIbeta subunits is the residue forming the disulfide bond with Cys-199 in the PKA catalytic alpha subunit. A new molecular point for cross-talk among heterologous signal transduction pathways is demonstrated.

1. Non-steroidal anti-inflammatory drugs (NSAIDs) [acetylsalicylic acid (ASS), naproxen, nimesulide and piroxicam] decreased adrenaline- or dibutyryl cAMP-stimulated glycerol release in isolated adipocytes. We aimed to determine the mechanism of this NSAIDs action. 2. Non-steroidal anti-inflammatory drugs decreased cAMP-dependent protein kinase A (PKA) activity in rat adipocyte lysates and in a commercial bovine heart PKA holoenzyme. If added before cAMP, NSAIDs impaired PKA activation by the cyclic nucleotide; however, if PKA was first activated by cAMP, NSAIDs were ineffective. NSAIDs were also ineffective against PKA catalytic subunits. 3. Consequently, NSAIDs lowered hormone-sensitive lipase translocation from cytosol to lipid storage droplets in adipocytes lysates, the critical event to promote lipolysis. 4. These results indicate that inhibition of PKA activation explains NSAIDs-induced decrease in adrenaline-stimulated lipolysis. We suggest that reproduction of such inhibition in nociceptive cells might enhance the understanding of the mechanism underlying the analgesic effects of NSAIDs.

Background Non-steroidal anti-inflammatory drugs (NSAIDs) —aspirin, naproxen, nimesulide, and piroxicam— lowered activation of type II cAMP-dependent protein kinase A (PKA-II) in isolated rat adipocytes, decreasing adrenaline- and dibutyryl cAMP (Bt2cAMP)-stimulated lipolysis. The molecular bases of insulin-like actions of NSAID were studied. Results Based on the reported inhibition of lipolysis by H2O2, catalase was successfully used to block NSAID inhibitory action on Bt2cAMP-stimulated lipolysis. NSAID, at (sub)micromolar range, induced an H2O2 burst in rat adipocyte plasma membranes and in whole adipocytes. NSAID-mediated rise of H2O2 was abrogated in adipocyte plasma membranes by: diphenyleneiodonium, an inhibitor of NADPH oxidase (NOX); the NOX4 antibody; and cytochrome c, trapping the NOX-formed superoxide. These three compounds prevented the inhibition of Bt2cAMP-stimulated lipolysis by NSAIDs. Inhibition of aquaporin-mediated H2O2 transport with AgNO3 in adipocytes allowed NOX activation but prevented the lipolysis inhibition promoted by NSAID: i.e., once synthesized, H2O2 must reach the lipolytic machinery. Since insulin inhibits adrenaline-stimulated lipolysis, the effect of aspirin on isoproterenol-stimulated lipolysis in rat adipocytes was studied. As expected, isoproterenol-mediated lipolysis was blunted by both insulin and aspirin. Conclusions NSAIDs activate NOX4 in adipocytes to produce H2O2, which impairs cAMP-dependent PKA-II activation, thus preventing isoproterenol-activated lipolysis. H2O2 signaling in adipocytes is a novel and important cyclooxygenase-independent effect of NSAID.