Activation of the mitogen-activated protein kinase cascade by pertussis toxin-sensitive and -insensitive pathways in cultured ventricular cardiomyocytes.

The involvement of pertussis toxin (PTX)-sensitive and -insensitive pathways in the activation of the mitogen-activated protein kinase (MAPK) cascade was examined in ventricular cardiomyocytes cultured from neonatal rats. A number of agonists that activate heterotrimeric G-protein-coupled receptors stimulated MAPK activity after exposure for 5 min. These included foetal calf serum (FCS), endothelin-1 (these two being the most effective of the agonists examined), phenylephrine, endothelin-3, lysophosphatidic acid, carbachol, isoprenaline and angiotensin II. Activation of MAPK and MAPK kinase (MEK) by carbachol returned to control levels within 30-60 min, whereas activation by FCS was more sustained. FPLC on Mono Q showed that carbachol and FCS activated two peaks of MEK and two peaks of MAPK (p42MAPK and p44MAPK). Pretreatment of cells with PTX for 24 h inhibited the activation of MAPK by carbachol, FCS and lysophosphatidic acid, but not that by endothelin-1, phenylephrine or isoprenaline. Involvement of G-proteins in the activation of the cardiac MAPK cascade was demonstrated by the sustained (PTX-insensitive) activation of MAPK (and MEK) after exposure of cells to AlF4-. AlF4- activated PtdIns hydrolysis, as did endothelin-1, endothelin-3, phenylephrine and FCS. In contrast, the effect of lysophosphatidic acid on PtdIns hydrolysis was small and carbachol was without significant effect even after prolonged exposure. We conclude that PTX-sensitive (i.e. Gi/G(o)-linked) and PTX-insensitive (i.e. Gq/Gs-linked) pathways of MAPK activation exist in neonatal ventricular myocytes. FCS may stimulate the MAPK cascade through both pathways.

Activation of p21-activated protein kinase alpha (alpha PAK) by hyperosmotic shock in neonatal ventricular myocytes.

The p21-activated protein kinases (PAKs) may participate in signalling from Cdc42/Rac1 to the stress-regulated MAPKs (SAPKs/JNKs and p38-/HOG-1-related-MAPKs). We characterized the expression and regulation of alpha PAK in cultured ventricular myocytes. alpha PAK was specifically immunoprecipitated from myocyte extracts. High basal alpha PAK activity was detected in unstimulated myocytes. Its activity was increased rapidly (<30 s) by hyperosmotic shock in the presence of okadaic acid, and was maximal by 3 min (187 +/- 7% relative to unstimulated cells). Endothelin-1 and interleukin-1beta, which also activate SAPKs/JNKs, did not increase alpha PAK activity and presumably act through different PAK isoforms or other mechanisms.

Activation of mitogen-activated protein kinases (p38-MAPKs, SAPKs/JNKs and ERKs) by adenosine in the perfused rat heart.

Adenosine and mitogen-activated protein kinases (MAPKs) have been separately implicated in cardiac ischaemic preconditioning. We investigated the activation of MAPK subfamilies by adenosine in perfused rat hearts. p38-MAPK was rapidly phosphorylated and activated (10-fold activation, maximal at 5 min) by 10 mM adenosine, as was the p38-MAPK substrate, MAPKAPK2 (4.5-fold). SAPKs/JNKs were activated (5-fold) and ERKs were phosphorylated (both maximal at 5 min). The concentration dependences of activation of p38-MAPK and ERKs were biphasic with a 'high affinity' component (maximal at 10-100 microM adenosine) and a 'low affinity' component that had not saturated at 10 mM. SAPKs/JNKs were activated only by 10 mM adenosine. These results are consistent with MAPK involvement in adenosine-mediated ischaemic preconditioning.

Prolonged exposure to palmitate impairs fatty acid oxidation despite activation of AMP-activated protein kinase in skeletal muscle cells

The aim of this study was to investigate the chronic effects of palmitate on fatty acid (FA) oxidation, AMPK/ACC phosphorylation/activation, intracellular lipid accumulation, and the molecular Mechanisms involved in these processes in skeletal muscle cells. Exposure of L6 myotubes for 8 h to 200, 400, 600, and 800 mu M of palmitate did rot affect cel viability but significantly reduced FA oxidation by similar to 26.5%, similar to 43.5%, similar to 50%, and similar to 47%, respectively. Interestingly, this occurred despite significant increases in AMPK (similar to 2.5-fold) and ACC (similar to 3-fold) phosphorylation and in malonyl-CoA decarboxylase activity (similar to 38-60%). Low concentrations of palmitate (50-100 mu M) caused an increase (similar to 30%) in CPT-I activity. However, as the concentration of palmitate increased, CPT-I activity decreased by similar to 32% after exposure for 8 h to 800 mu M of palmitate. Although FA uptake was reduced (similar to 35%) in cells exposed to increasing, palmitate concentrations, intracellular lipid accumulation increased in a dose-dependent manner, reaching values similar to 2.3-, similar to 3-, and 4-fold higher than control in muscle cells exposed to 400, 600, and 800 mu M palmitate, respectively. Interestingly, myotubes exposed to 400 mu M of palmitate for 1h increased basal glucose uptake and glycogen synthesis by similar to 40%. However, as time of incubation in the presence of palmitate progressed from 1 to 8h, these increases were abolished and a time-dependent inhibition of insulin-stimulated glucose uptake (similar to 65%) and glycogen synthesis (30%) was observed in myotubes. These findings may help explain the dysfunctional adaptations that occur in glucose and FA Metabolism in skeletal muscle under conditions of chronically elevated circulating levels of non-esterified FAs. Such as in obesity and Type 2 Diabetes.

Endurance training activates AMP-activated protein kinase, increases expression of uncoupling protein 2 and reduces insulin secretion from rat pancreatic islets

Endurance exercise is known to enhance peripheral insulin sensitivity and reduce insulin secretion. However, it is unknown whether the latter effect is due to the reduction in plasma substrate availability or alterations in beta-cell secretory machinery. Here, we tested the hypothesis that endurance exercise reduces insulin secretion by altering the intracellular energy-sensitive AMP-activated kinase (AMPK) signaling pathway. Male Wistar rats were submitted to endurance protocol training one, three, or five times per week, over 8 weeks. After that, pancreatic islets were isolated, and glucose-induced insulin secretion (GIIS), glucose transporter 2 (GLUT2) protein content, total and phosphorylated calmodulin kinase kinase (CaMKII), and AMPK levels as well as peroxisome proliferator-activated receptor-gamma coactivator-1-alpha (PGC-1 alpha) and uncoupling protein 2 (UCP2) content were measured. After 8 weeks, chronic endurance exercise reduced GIIS in a dose-response manner proportionally to weekly exercise frequency. Contrariwise, increases in GLUT2 protein content, CaMKII and AMPK phosphorylation levels were observed. These alterations were accompanied by an increase in UCP2 content, probably mediated by an enhancement in PGC-1 alpha protein expression. In conclusion, chronic endurance exercise induces adaptations in beta-cells leading to a reduction in GIIS, probably by activating the AMPK signaling pathway. Journal of Endocrinology (2011) 208, 257-264

Metabolic oxidative stress elicited by the copper(II) complex [Cu(isaepy)(2)] triggers apoptosis in SH-SY5Y cells through the induction of the AMP-activated protein kinase/p38(MAPK)/p53 signalling axis: evidence for a combined use with 3-bromopyruvate in neuroblastoma treatment

We have demonstrated previously that the complex bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N`]copper(II), named [Cu(isaepy)(2)], induces AMPK (AMP-activated protein kinase)-dependent/p53-mediated apoptosis in tumour cells by targeting mitochondria. In the present study, we found that p38(MAPK) (p38 mitogen-activated protein kinase) is the molecular link in the phosphorylation cascade connecting AMPK to p53. Transfection of SH-SY5Y cells with a dominant-negative mutant of AMPK resulted in a decrease in apoptosis and a significant reduction in phospho-active p38(MAPK) and p53. Similarly, reverse genetics of p38(MAPK) yielded a reduction in p53 and a decrease in the extent of apoptosis, confirming an exclusive hierarchy of activation that proceeds via AMPK/p38(MAPK)/p53. Fuel supplies counteracted [Cu(isaepy)(2)]-induced apoptosis and AMPK/p38(MAPK)/p53 activation, with glucose being the most effective, suggesting a role for energetic imbalance in [Cu(isaepy)(2)] toxicity. Co-administration of 3BrPA (3-bromopyruvate), a well-known inhibitor of glycolysis, and succinate dehydrogenase, enhanced apoptosis and AMPK/p38(MAPK)/p53 signalling pathway activation. Under these conditions, no toxic effect was observed in SOD (superoxide dismutase)-overexpressing SH-SY5Y cells or in PCNs (primary cortical neurons), which are, conversely, sensitized to the combined treatment with [Cu(isaepy)(2)] and 3BrPA only if grown in low-glucose medium or incubated with the glucose-6-phosphate dehydrogenase inhibitor dehydroepiandrosterone. Overall, the results suggest that NADPH deriving from the pentose phosphate pathway contributes to PCN resistance to [Cu(isaepy)(2)] toxicity and propose its employment in combination with 3BrPA as possible tool for cancer treatment.

The isatin-Schiff base copper(II) complex Cu(isaepy)(2) acts as delocalized lipophilic cation, yields widespread mitochondrial oxidative damage and induces AMP-activated protein kinase-dependent apoptosis

We previously demonstrated that Bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl) pyridine-N, N`] copper(II) [Cu(isaepy)(2)] was an efficient inducer of the apoptotic mitochondrial pathway. Here, we deeply dissect the mechanisms underlying the ability of Cu(isaepy)(2) to cause mitochondriotoxicity. In particular, we demonstrate that Cu(isaepy)(2) increases NADH-dependent oxygen consumption of isolated mitochondria and that this phenomenon is associated with oxy-radical production and insensitive to adenosine diphosphate. These data indicate that Cu(isaepy)(2) behaves as an uncoupler and this property is also confirmed in cell systems. Particularly, SH-SY5Y cells show: (i) an early loss of mitochondrial transmembrane potential; (ii) a decrease in the expression levels of respiratory complex components and (iii) a significant adenosine triphosphate (ATP) decrement. The causative energetic impairment mediated by Cu(isaepy)(2) in apoptosis is confirmed by experiments carried out with rho(0) cells, or by glucose supplementation, where cell death is significantly inhibited. Moreover, gastric and cervix carcinoma AGS and HeLa cells, which rely most of their ATP production on oxidative phosphorylation, show a marked sensitivity toward Cu(isaepy)(2). Adenosine monophosphate-activated protein kinase (AMPK), which is activated by events increasing the adenosine monophosphate: ATP ratio, is deeply involved in the apoptotic process because the overexpression of its dominant/negative form completely abolishes cell death. Upon glucose supplementation, AMPK is not activated, confirming its role as fuel-sensing enzyme that positively responds to Cu(isaepy)(2)-mediated energetic impairment by committing cells to apoptosis. Overall, data obtained indicate that Cu(isaepy)(2) behaves as delocalized lipophilic cation and induces mitochondrial-sited reactive oxygen species production. This event results in mitochondrial dysfunction and ATP decrease, which in turn triggers AMPK-dependent apoptosis.

Thrombomodulin-independent activation of protein C and specificity of hemostatically active snake venom serine proteinases - Crystal structures of native and inhibited Agkistrodon contortrix contortrix protein C activator

Protein C activation initiated by the thrombin-thrombomodulin complex forms the major physiological anticoagulant pathway. Agkistrodon contortrix contortrix protein C activator, a glycosylated single-chain serine proteinase, activates protein C without relying on thrombomodulin. The crystal structures of native and inhibited Agkistrodon contortrix contortrix protein C activator determined at 1.65 and 1.54 angstrom resolutions, respectively, indicate the pivotal roles played by the positively charged belt and the strategic positioning of the three carbohydrate moieties surrounding the catalytic site in protein C recognition, binding, and activation. Structural changes in the benzamidine-inhibited enzyme suggest a probable function in allosteric regulation for the anion-binding site located in the C-terminal extension, which is fully conserved in snake venom serine proteinases, that preferentially binds Cl1- instead of SO42-.

Crystallization and preliminary X-ray crystallographic studies of Protac (R), a commercial protein C activator isolated from Agkistrodon contortrix contortrix venom

The protein C pathway plays an important role in the control and regulation of the blood coagulation cascade and prevents the propagation of the clotting process on the endothelium surface. In physiological systems, protein C activation is catalyzed by thrombin, which requires thrombomodulin as a cofactor. The protein C activator from Agkistrodon contortrix contortrix acts directly on the zymogen of protein C converting it into the active form, independently of thrombomodulin. Suitable crystals of the protein C activator from Agkistrodon contortrix contortrix were obtained from a solution containing 2 M ammonium sulfate as the precipitant and these crystals diffracted to 1.95 angstrom resolution at a synchrotron beamline. The crystalline array belongs to the monoclinic space group C2 with unit cell dimensions a=80.4, b = 63.3 and c = 48.2 angstrom, alpha = gamma = 90.0 degrees and beta = 90.8 degrees. (C) 2005 Elsevier B.V. All rights reserved.

Exercise training decreases mitogen-activated protein kinase phosphatase-3 expression and suppresses hepatic gluconeogenesis in obese mice

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Silencing mitogen-activated protein kinase-activated protein kinase-2 arrests inflammatory bone loss

p38 mitogen-activated protein kinases (MAPKs) are critical for innate immune signaling and subsequent cytokine expression in periodontal inflammation and bone destruction. In fact, previous studies show that systemic p38 MAPK inhibitors block periodontal disease progression. However, development of p38 MAPK inhibitors with favorable toxicological profiles is difficult. Here, we report our findings regarding the contribution of the downstream p38 MAPK substrate, mitogen-activated protein kinase-activated protein kinase 2 (MK2 or MAPKAPK-2), in immune response modulation in an experimental model of pathogen-derived lipopolysaccharide (LPS)-induced periodontal bone loss. To determine whether small interfering RNA (siRNA) technology has intraoral applications, we initially validated MK2 siRNA specificity. Then, gingival tissue surrounding maxillary molars of rats was injected with MK2 siRNA or scrambled siRNA at the palatal regions of bone loss. Intraoral tissues treated with MK2 siRNA had significantly less MK2 mRNA expression compared with scrambled siRNA-treated tissues. MK2 siRNA delivery arrested LPS-induced inflammatory bone loss, decreased inflammatory infiltrate, and decreased osteoclastogenesis. This proof-of-concept study suggests a novel target using an intraoral RNA interference strategy to control periodontal inflammation.