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The microbial unification of the world Unbeknownst to many, we are living during the seventh pandemic of cholera in modern times. Descriptions of a cholera-like illness in India go back [...]

1. Introduction Chronic heart failure (CHF) is a global health problem that is the end-stage of multiple heart diseases, such as hypertension, coronary heart disease, and cardiomyopathy. CHF patients are [...], Most sudden cardiac death in chronic heart failure (CHF) is caused by malignant ventricular arrhythmia (VA); however, the molecular mechanism remains unclear. This study aims to explore the effect of exchange proteins directly activated by cAMP (Epac) on VA in CHF and the potential molecular mechanism. Transaortic constriction was performed to prepare CHF guinea pigs. Epac activation model was obtained with 8-pCPT administration. Programmed electrical stimulation (PES) was performed to detect effective refractory period (ERP) or induce VA. Isolated adult cardiomyocytes were treated with 8-pCPT and (or) the Epac inhibitor. Cellular electrophysiology was examined by whole-cell patch clamp. With Epac activation, corrected QT duration was lengthened by 12.6%. The 8-pCPT increased action potential duration (APD) ([APD.sub.50]: 236.9 [+ or -] 18.07 ms vs. 328.8 [+ or -] 11.27 ms, p < 0.05; [APD.sub.90]: 264.6 [+ or -] 18.22 ms vs. 388.6 [+ or -] 6.47 ms, p < 0.05) and decreased rapid delayed rectifier potassium ([I.sub.Kr]) current (tail current density: I. 1 [+ or -] 0.08 pA/pF vs. 0.7 [+ or -] 0.03 pA/pF, p < 0.05). PES induced more malignant arrhythmias in the 8-pCPT group than in the control group (3/4 vs. 0/8, p < 0.05). The selective Epac1 inhibitor CE3F4 rescued the drop in [I.sub.Kr] after 8-pCPT stimulation (tail current density: 0.5 [+ or -] 0.02 pA/pF vs. 0.6 [+ or -] 0.03 pA/pF, p < 0.05). In conclusion, Epac1 regulates [I.sub.Kr], APD, and ERP in guinea pigs, which could contribute to the proarrhythmic effect of Epac1 in CHF. Key words: exchange proteins directly activated by cAMP, rapid delayed rectifier potassium, repolarization, chronic heart failure, ventricular arrhythmia. Dans l&apos;insuffisance cardiaque chronique (ICC), la plupart des morts subites sont causees par des arythmies ventriculaires (AV) malignes. Cependant, les modes d&apos;action moleculaires demeurent a clarifier. Cette etude a pour but d&apos;explorer l&apos;effet des proteines d&apos;echange activees directement par l&apos;AMPc (Epac pour << exchange proteins directly activated by cAMP >>) sur les AV dans l&apos;ICC ainsi que le mode d&apos;action moleculaire eventuel. Nous avons procede a une constriction transaortique en vue d&apos;obtenir des cobayes atteints d&apos;ICC. Nous avons obtenu le modele d&apos;activation des Epac par l&apos;administration de 8-pCPT. Nous avons utilise la stimulation electrique programmee (SEP) en vue d&apos;etablir la periode refractive effective (PRE) ou de provoquer des AV. Nous avons mis des cardiomyocytes adultes isoles en presence de 8-pCPT et/ou de l&apos;inhibiteur des Epac. L&apos;electrophysiologie cellulaire a ete etudiee a l&apos;aide de la technique de << patch clamp >> sur cellules entieres. Avec l&apos;activation des Epac, la duree du segment QT corrigee (QTc) se prolongeait de 12,6 %. Le 8-pCPT entrainait une augmentation de la duree du potentiel d&apos;action (DPA) (DPA50 : 236,9 [+ or -] 18,07 ms vs 328,8 [+ or -] II, 27 ms, p < 0,05; DPA90 : 264,6 [+ or -] 18,22 ms vs 388,6 [+ or -] 6,47 ms, p < 0,05) avec une diminution du courant [I.sub.Kr] (densite de courant de queue : 1,1 [+ or -] 0,08 pA/pF vs 0,7 [+ or -] 0,03 pA/pF, p < 0,05). L&apos;ESP a entraine plus d&apos;arythmies malignes dans le groupe 8-pCPT que chez les temoins (3/4 vs 0/8, p < 0,05). Le CE3F4, un inhibiteur selectif des Epac1, permettait de prevenir l&apos;abaissement d&apos;[I.sub.Kr] apres la stimulation par le 8-pCPT (densite de courant de queue : 0,5 [+ or -] 0,02 pA/pF vs 0,6 [+ or -] 0,03 pA/pF, p < 0,05). En conclusion, les Epac1 participent a la regulation du courant [I.sub.Kr], de la DPA et de la PRE chez le cobaye, ce qui pourrait contribuer a l&apos;effet proarythmique des Epac1 dans l&apos;ICC. [Traduit par la Redaction] Mots-cles : proteines d&apos;echange activees directement par l&apos;AMPc, [I.sub.Kr], repolarisation, insuffisance cardiaque chronique, arythmie ventriculaire.

Introduction Metabolic syndrome (MetS) represents a group of interrelated risk factors that predict cardiovascular disease and increased resistance to the effects of insulin, among others (Roberts et al. 2013; Gluvic [...], Metabolic syndrome (MetS) is a cluster of risk factors, including insulin resistance among others, underlying the development of diabetes and (or) cardiovascular diseases. Studies show a close relationship between cardiac dysfunction and abnormal cAMP catabolism, which contributes to pathological remodelling. Stimulating the synthesis of cAMP via suppression of phosphodiesterases (PDEs) has positive therapeutic effects. Therefore, we examined the role of PDEs on cardiac dysfunction in high-carbohydrate diet-induced MetS rats. We first demonstrated significantly high expression levels of PDE3 and PDE4, the most highly expressed subtypes, together with depressed cAMP levels in heart tissue from MetS rats. Second, we demonstrated the activity of these PDEs by using either their basal or PDE inhibitor-induced intracellular levels of cAMP and [Ca.sup.2+], the transient intracellular [Ca.sup.2+] changes under electrical stimulation, isometric contractions in papillary muscle strips and some key signalling proteins (such as RyR2, PLN, PP1A, and PKA) are responsible for the [Ca.sup.2+] homeostasis in isolated cardiomyocytes from MetS rats. The clear recovery in decreased basal cAMP levels, increased protein expression levels of PDE3 and PDE4, and positive responses in the altered [Ca.sup.2+] homeostasis to PDE inhibitors as seen in our study can provide important insights about the roles of activated PDEs in depressed contractile activity in hearts from MetS rats. Key words: heart function, calcium transients, insulin resistance, high-sucrose diet, prediabetes, PDE3, PDE4. Le syndrome metabolique consiste en un regroupement de facteurs de risque, y compris la resistance a l'insuline, qui sous-tendent l'apparition du diabete ou des maladies cardiovasculaires. Les etudes montrent la presence d'un lien etroit entre le dysfonctionnement cardiaque et le catabolisme anormal de l'AMPc, ce qui contribue au remodelage pathologique. La stimulation de la synthese de l'AMPc par l'intermediaire de l'inhibition des phosphodiesterases (PDE) a des effets therapeutiques positifs. Par consequent, nous avons examine le role des PDE dans le dysfonctionnement cardiaque chez des rats auxquels nous administrions un regime a haute teneur en glucides en vue de provoquer un syndrome metabolique. Nous avons tout d'abord observe une nette augmentation des taux d'expression de la PDE3 et de la PDE4 (les sous-types les plus exprimes) avec un abaissement des taux d'AMPc dans le tissu cardiaque de rats presentant un syndrome metabolique. Ensuite, nous avons etabli clairement l'activite de ces PDE en etudiant soit les taux intracellulaires d'AMPc et de [Ca.sup.2+] a l'etat basal ou en reaction a des inhibiteurs de la PDE, soit les modifications transitoires du [Ca.sup.2+] intracellulaire avec la stimulation electrique, soit des contractions isometriques dans des bandelettes de muscle papillaire, et certaines proteines de signalisation cles (comme RyR2, PLN, PP1A et PKA) responsables de l'homeostasie du [Ca.sup.2+] dans des cardiomyocytes isoles de rats presentant un syndrome metabolique. Un net retablissement des taux d'AMPc precedemment abaisses, une augmentation des taux d'expression en proteines de la PDE3 et de la PDE4 et des reactions favorables contre la deterioration de l'homeostasie du [Ca.sup.2+] par des inhibiteurs des PDE comme le montre notre etude apportent des renseignements importants quant aux roles des PDE activees dans la diminution de l'activite contractile du coeur de rats presentant un syndrome metabolique. [Traduit par la Redaction] Mots-cles : fonction cardiaque, courants calciques transitoires, resistance a l'insuline, regime alimentaire a haute teneur en saccharose, prediabete, PDE3, PDE4.

Introduction About 3.3 million deaths (5.9% of all deaths) were attributed to alcohol consumption in 2012 according to the 2014 WHO Global Report (Collart et al. 2015). Alcoholic liver disease [...], Hepatic stellate cells (HSCs) activation represents an essential event during alcoholic liver fibrosis (ALF). Previous studies have demonstrated that the rat HSCs could be significantly activated after exposure to 200 µmol/L acetaldehyde for 48 h, and the cAMP/PKA signaling pathways were also dramatically upregulated in activated HSCs isolated from alcoholic fibrotic rat liver. Exchange protein activated by cAMP (EPAC) is a family of guanine nucleotide exchange factors (GEFs) for the small Ras-like GTPases Rap, and is being considered as a vital mediator of cAMP signaling in parallel with the principal cAMP target protein kinase A (PKA). Our data showed that both cAMP/PKA and cAMP/EPAC signaling pathways were involved in acetaldehyde- induced HSCs. Acetaldehyde could reduce the expression of EPAC1 while enhancing the expression of EPAC2. The cAMP analog Me- cAMP, which stimulates the EPAC/Rap1 pathway, could significantly decrease the proliferation and collagen synthesis of acetaldehyde-induced HSCs. Furthermore, depletion of EPAC2, but not EPAC1, prevented the activation of HSC measured as the production of α-SMA and collagen type I and III, indicating that EPAC1 appears to have protective effects on acetaldehyde- induced HSCs. Curiously, activation of PKA or EPAC perhaps has opposite effects on the synthesis of collagen and α-SMA: EPAC activation by Me-cAMP increased the levels of GTP-bound (activated) Rap1 while PKA activation by Phe-cAMP had no significant effects on such binding. These results suggested that EPAC activation could inhibit the activation and proliferation of acetaldehyde- induced HSCs via Rap1. Key words: acetaldehyde, EPAC1, EPAC2, hepatic stellate cells, Rap1. L'activation des cellules de Kupffer correspond a un facteur essentiel de l'evolution de la fibrose hepatique alcoolique. Des etudes anterieures ont montre que les cellules de Kupffer de rat pouvaient etre activees de facon marquee apres l'exposition a de l'acetaldehyde a 200 µmol/L pendant 48 h. Les voies de signalisation AMPc-PKA (adenosine monophosphate cyclique proteine kinase A) etaient aussi nettement regulees a la hausse dans les cellules de Kupffer activees isolees de foie de rats presentant une fibrose alcoolique. Par ailleurs, la proteine d'echange activee par l'AMPc (EPAC) est une famille de facteurs d'echange de la guanine (nucleotide) pour les petites GTPases Rap apparentees aux proteines Ras, et on la considere comme un mediateur essentiel de la voie de signalisation de l'AMPc en parallele avec la PKA, principale cible de l'AMPc. Nos donnees ont montre que les voies de signalisation AMPc-PKA et AMPc-EPAC jouaient toutes deux un role dans l'induction des cellules de Kupffer par l'acetaldehyde. En fait, l'acetaldehyde pourrait faire diminuer l'expression de la proteine EPAC1 tout en faisant augmenter l'expression de la proteine EPAC2. Par ailleurs, la Me-AMPc, un analogue de l'AMPc qui stimule la voie de signalisation EPAC-Rap1, pourrait attenuer de facon marquee la proliferation des cellules de Kupffer induites par l'acetaldehyde ainsi que la synthese du collagene dont elles sont responsables. De plus, la diminution des taux de proteine EPAC2, mais pas celle des taux de la proteine EPAC1, a empeche l'activation des cellules de Kupffer (evaluee selon la production d'actine a des cellules musculaires lisses et de collagene de types I et III), ce qui indique que la proteine EPAC1 pourrait avoir des effets protecteurs contre les cellules de Kupffer induites par l'acetaldehyde. Curieusement, l'activation de la PKA et de la proteine EPAC ont peut-etre des effets opposes sur la synthese de collagene et d'actine a des cellules musculaires lisses : l'activation de la proteine EPAC par la Me-AMPc entrainait une augmentation des taux de proteine Rap1 liee au GTP (activee), alors que l'activation de la PKA par la Phe-AMPc n'avait aucun effet notable sur cette liaison. Ces resultats laissent entendre que l'activation de la proteine EPAC pourrait inhiber l'activation et la proliferation des cellules de Kupffer induites par l'acetaldehyde par l'intermediaire de la proteine Rap1. [Traduit par la Redaction] Mots-cles : acetaldehyde, EPAC1, EPAC2, cellules de Kupffer, Rap1.

Quantitative mass spectrometry was used to identify hormone-dependent signaling pathways in renal medullary thick ascending limb (mTAL) cells via phosphoproteomic analysis. Active transport of NaCI across the mTAL epithelium is accelerated by hormones that increase cAMP levels (vasopressin, glucagon, parathyroid hormone, and calcitonin), mTAL suspensions from rat kidneys were exposed (15 min) to a mixture of these four hormones. Tryptic phosphopeptides (immobilized metal affinity chromatography-enriched) were identified and quantified by mass spectrometry (LTQ-Orbitrap) using label-free methodology. We quantified a total of 654 phosphopeptides, of which 414 were quantified in three experimental pairs (hormone vs. vehicle). Of these phosphopeptides, 82% were statistically unchanged in abundance in response to the hormone mixture. In contrast, 48 phosphopeptides were significantly increased, whereas 28 were significantly decreased. The population of up-regulated phosphopeptides was highly enriched in basophilic kinase substrate motifs (AGC or calmodulin-sensitive kinase families), whereas the down-regulated sites were dominated by 'proline-directed' motifs (cyclin-dependent or MAP kinase families). Bioinformatic classification uncovered overrepresentation of transmembrane transporters, protein phosphatase regulators, and cytoskeletal binding proteins among the regulated proteins: Immunoblotting with phospho-specific antibodies confirmed cAMP/vasopressin-dependent phosphorylation at Thr96, Ser126, and Ser874 of the [Na.sup.+]:[K.sup.+]:2[CI.sup.-] cotransporter NKCC2, at Ser552 of the [Na.sup.+]:[H.sup.+] exchanger NHE3, and at Ser552 of [beta]-catenin. Vasopressin also increased phosphorylation of NKCC2 at both Ser126 (more than fivefold) and Ser874 (more than threefold) in rats in vivo. Both sites were phosphorylated by purified protein kinase A during in vitro assays. These results support the view that, although protein kinase A plays a central role in mTAL signaling, additional kinases, including those that target proline-directed motifs, may be involved. protein phosphatase | glucose transporters | mass spectrometry | ion transporters | protein kinase doi/ 10.1073/pnas.1007424107

The [[beta].sub.1]-and [[beta].sub.2]-adrenergic receptors ([beta]ARs) on the surface of cardiomyocytes mediate distinct effects on cardiac function and the development of heart failure by regulating production of the second messenger cyclic adenosine monophosphate (cAMP). The spatial localization in cardiomyocytes of these [beta]ARs, which are coupled to heterotrimeric guanine nucleotide--binding proteins (G proteins), and the functional implications of their localization have been unclear. We combined nanoscale live-cell scanning ion conductance and fluorescente resonance energy transfer microscopy techniques and found that, in cardiomyocytes from healthy adult rats and mice, spatially confined [[beta].sub.2]AR-induced cAMP signals are localized exclusively to the deep transverse tubules, whereas functional [[beta].sub.1]ARs are distributed across the entire cell surface. In cardiomyocytes derived from a rat model of chronic heart failure, [[beta].sub.2]ARs were redistributed from the transverse tubules to the cell crest, which led to diffuse receptor-mediated cAMP signaling. Thus, the redistribution of [[beta].sub.2]ARs in heart failure changes compartmentation of cAMP and might contribute to the failing myocardial phenotype. 10.1126/science.1185988

Melanoma, the most malignant form of human skin cancer, has a poor prognosis due to its strong metastatic ability. It was recently demonstrated that Epac, an effector molecule of cAMP, is involved in regulating cell migration; however, the role of Epac in melanoma cell migration remains unclear. We thus examined whether Epac regulates cell migration and metastasis of melanoma. Epac activation, by either specific agonist or overexpression of Epac, increased melanoma cell migration. Deletion of endogenous Epac with small interfering RNA decreased basal melanoma cell migration. These data suggested a major role of Epac in melanoma cell migration. Epac-induced cell migration was mediated by translocation of syndecan-2, a cell-surface heparan sulfate proteoglycan, to lipid rafts. This syndecan-2 translocation was regulated by tubulin polymerization via the Epac/phosphoinositol-3 kinase pathway. Epac-induced cell migration was also regulated by the production of heparan sulfate, a major extracellular matrix. Epac-induced heparan sulfate production was attributable to the increased expression of N-deacetylase/N-sulfotransferase-1 (NDST-1) accompanied by an increased NDST-1 translation rate. Finally, Epac overexpression enhanced lung colonization of melanoma cells in mice. Taken together, these data indicate that Epac regulates melanoma cell migration/ metastasis mostly via syndecan-2 translocation and heparan sulfate production. metastasis; phosphoinositol-3 kinase; N-deacetylase/N-sulfotransferase-1 doi: 10.1152/ajpcell.00129.2009

Mitosis is triggered by activation of Cdk1, a cyclin-dependent kinase. Conserved checkpoint mechanisms normally inhibit Cdk1 by inhibitory phosphorylation during interphase, ensuring that DNA replication and repair is completed before cells begin mitosis. In metazoans, this regulatory mechanism is also used to coordinate cell division with critical developmental processes, such as cell invagination. Two types of Cdk1 inhibitory kinases have been found in metazoans. They differ in subcellular localization and Cdk1 target-site specificity: one (Weel) being nuclear and the other (Myt1), membrane-associated and cytoplasmic. Drosophila has one representative of each: dMyt1 and dwee1. Although dwee1 and dMyt1 are not essential for zygotic viability, loss of both resulted in synthetic lethality, indicating that they are partially functionally redundant. Bristle defects in myt1 mutant adult flies prompted a phenotypic analysis that revealed cell-cycle defects, ectopic apoptosis, and abnormal responses to ionizing radiation in the myt1 mutant imaginal wing discs that give rise to these mechanosensory organs. Cdk1 inhibitory phosphorylation was also aberrant in these mill mutant imaginal wing discs, indicating that dMyt1 serves Cdk1 regulatory functions that are important both for normal cell-cycle progression and for coordinating mitosis with critical developmental processes.

The goal of this study was to identify a mechanism regulating cholesterol accumulation in cystic fibrosis (CF) cells. Both CFTR activation and expression are regulated by the cAMP pathway, and it is hypothesized that a feedback response involving this pathway may be involved in the phenotype of cholesterol accumulation. To examine the role of the cAMP pathway in cholesterol accumulation, we treated two CF model cell lines with the Rp diastereomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS) and visualized by filipin staining. Rp-cAMPS treatment eliminated cholesterol accumulation in CF cells, whereas 8-bromo-cAMP treatment led to cholesterol accumulation in wild-type cells. To confirm these findings in an independent model system, we also examined the role of cAMP in modulating cholesterol accumulation in Niemann-Pick type C (NPC) fibroblasts. Expression of the protein related to NPC, NPC1, is also directly regulated by cAMP; therefore, it is postulated that NPC cells exhibit the same cAMP-mediated control of cholesterol accumulation. Cholesterol accumulation in NPC cells also was reduced by the presence of Rp-cAMPS. Expression of [beta]-arrestin-2 ([beta]arr2), a marker of cellular response to cAMP signaling, was significantly elevated in CF model cells, [Cftr.sup.-/-] MNE, primary tissue obtained by nasal scrapes from CF subjects, and in NPC fibroblasts compared with respective controls. Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate

Several stress conditions are characterized by activation of 5'-AMP-activated protein kinase (AMPK) and the development of leucine resistance in skeletal muscle. In the present study, we determined whether direct activation of the AMPK by 5-aminoimidazole-4-carboxamide-1-[beta]-D-ribonucleoside (AICAR) prevents the characteristic leucine-induced increase in protein synthesis by altering mammalian target of rapamycin (mTOR) signal transduction. Rats were injected with AICAR or saline (Sal) and 1 h thereafter received an oral gavage of leucine (or Sal). Efficacy of AICAR was verified by increased AMPK phosphorylation. AICAR decreased basal in vivo muscle (gastrocnemius) protein synthesis and completely prevented the leucine-induced increase, independent of a change in muscle adenine nucleotide concentration. AICAR also prevented the hyperphosphorylation of eukaryotic initiation factor (elF) 4E binding protein (4E-BP1), ribosomal protein S6 kinase (S6K1), S6, and elF4G in response to leucine, suggesting a decrease in mTOR activity. Moreover, AICAR prevented the leucine-induced redistribution of elF4E from the inactive elF4E x 4E-BP1 to the active elF4E x elF4G complex. This ability of AICAR to produce muscle leucine resistance could not be attributed to a change in phosphorylation of tuberous sclerosis complex (TSC)2, the formation of a TSC1 x TSC2 complex, the binding of raptor with mTOR, or the phosphorylation of eukaryotic elongation factor-2. However, the inhibitory actions of AICAR were associated with reduced phosphorylation of proline- rich Akt substrate-40 and increased phosphorylation of raptor, which represent potential mechanisms by which AICAR might be expected to inhibit leucine-induced increases in mTOR activity and protein synthesis under in vivo conditions.

Cold increases cutaneous vasoconstriction by unmasking the contractile activity of [[alpha].sub.2]c-adrenoceptors ([[alpha].sub.2]c-ARs) in vascular smooth muscle cells (VSMCs), which is mediated by the cold-induced mobilization of [[alpha].sub.2]c-ARs from the transGolgi to the cell surface. The expression of [[alpha].sub.2]c-ARs in human cutaneous VSMCs is under dual regulation by cyclic AMP: gene transcription is inhibited by cyclic AMP acting through protein kinase A but is increased by cyclic AMP acting through the exchange protein directly activated by cyclic AMP (EPAC) and the GTP-binding protein Rap 1. Experiments were performed to further characterize the Rap1 signaling pathway. Forskolin (10 [micro]M), the selective EPAC activator, 8-pCPT-2'-O-Me-cyclic AMP (CMC; 100 [micro]M), or a constitutively active mutant of Rap1 (Rap1CA) increased the activity of c-Jun N[H.sub.2]-terminal kinase (JNK) in human cutaneous VSMCs. This was associated with the increased phosphorylation of c-Jun and activation of an activator protein (AP)-1 reporter construct, which were inhibited by the JNK inhibitor SP600125 (3 [micro]M). Rap1CA increased the activity of an [[alpha].sub.2]c-AR promoter-reporter construct, which was inhibited by SP600125 (3 [micro]M) or by the mutation of an AP-1 binding site in the [[alpha].sub.2]c-AR promoter. Furthermore, forskolin (10 [micro]M) or CMC (100 [micro]M) increased the expression of the [[alpha].sub.2]c-AR protein, and these effects were inhibited by SP600125 (3 [micro]M). Therefore, cyclic AMP increases the expression of [[alpha].sub.2]c-ARs in cutaneous VSMCs by activating a novel Rap1 signaling pathway, mediated by the activation of JNK, AP-1, and the subsequent transcriptional activation of the [[alpha].sub.2]c-AR gene. By increasing the expression of cold-responsive [[alpha].sub.2]c-ARs, this pathway may contribute to enhanced cold-induced vasoconstriction in the cutaneous circulation, including Raynaud's phenomenon. Raynaud's phenomenon; activator protein-1; c-Jun N[H.sub.2]-terminal kinase; transcription; adenosine 5'-monophosphate

Although mutations in the [gamma]-subunit of AMP-activated protein kinase (AMPK) can result in excessive glycogen accumulation and cardiac hypertrophy, the mechanisms by which this occurs have not been well defined. Because >65% of cardiac AMPK activity is associated with the [gamma]1-subunit of AMPK, we investigated the effects of expression of an AMPK-activating [gamma]1-subunit mutant ([gamma]1 R70Q) on regulatory pathways controlling glycogen accumulation and cardiac hypertrophy in neonatal rat cardiac myocytes. Whereas expression of [gamma]1 R70Q displayed the expected increase in palmitate oxidation rates, rates of glycolysis were significantly depressed. In addition, glycogen synthase activity was increased in cardiac myocytes expressing [gamma]1 R70Q, due to both increased expression and decreased phospborylation of glycogen synthase. The inhibition of glycolysis and increased glycogen synthase activity were correlated with elevated glycogen levels in [gamma]1 R70Q-expressing myocytes. In association with the reduced phosphorylation of glycogen synthase, glycogen synthase kinase (GSK)-3[beta] protein and mRNA levels were profoundly decreased in the [gamma]1 R70Q-expressing myocytes. Consistent with GSK-3[beta] negatively regulating hypertrophy via inhibition of nuclear factor of activated T cells (NFAT), the dramatic downregulation of GSK-3[beta] was associated with increased nuclear activity of NFAT. Together, these data provide important new information about the mechanisms by which a mutation in the [gamma]-subunit of AMPK causes altered AMPK signaling and identify multiple pathways involved in regulating both cardiac myocyte metabolism and growth that may contribute to the development of the [gamma] mutant-associated cardiomyopathy. adenosine 5'-monophosphate-activated protein kinase; glycogen; metabolism; glycogen synthase kinase-3[beta]

5'-AMP-activated protein kinase (AMPK), by way of its inhibition of acetyl-CoA carboxylase (ACC), plays an important role in regulating malonyl-CoA levels and the rate of fatty acid oxidation in skeletal and cardiac muscle. In these tissues, LKB1 is the major AMPK kinase and is therefore critical for AMPK activation. The purpose of this study was to determine how the lack of muscle LKB1 would affect malonyl-CoA levels and/or fatty-acid oxidation. Comparing wild-type (WT) and skeletal/cardiac muscle-specific LKB1 knockout (KO) mice, we found that the 5-aminoimidazole-4-carboxamide-1-[beta]-D-ribofuranoside (AICAR)-stimulated decrease in malonyl-CoA levels in WT heart and quadriceps muscles was entirely dependent on the presence of LKB1, as was the AICAR-induced increase in fatty-acid oxidation in EDL muscles in vitro, since these responses were not observed in KO mice. Likewise, the decrease in malonyl-CoA levels after muscle contraction was attenuated in KO gastrocnemius muscles, suggesting that LKB1 plays an important role in promoting the inhibition of ACC, likely by activation of AMPK. However, since ACC phosphorylation still increased and malonyl-CoA levels decreased in KO muscles (albeit not to the levels observed in WT mice), whereas AMPK phosphorylation was entirely unresponsive, LKB1/AMPK signaling cannot be considered the sole mechanism for inhibiting ACC during and after muscle activity. Regardless, our results suggest that LKB1 is an important regulator of malonyl-CoA levels and fatty acid oxidation in skeletal muscle. 5'-AMP-activated protein kinase; acetyl-coenzyne A carboxylase; 5-aminoimidazole-4-carboxamide-1-[beta]-D-ribofuranoside; electric stimulation; muscle contraction

Hypothalamic hormones, including dopamine, regulate critical functions of pituitary cells via the cAMP-protein kinase A (PKA) pathway. The PKA-downstream transcription factor cAMP response element (CRE)-binding protein (CREB) is an integrating molecule that is also activated by many other protein kinase pathways. We investigated the involvement of CREB in the regulation of cell proliferation and the PRL promoter of rat lactotrophs in primary cell culture. Recombinant adenoviruses were used for efficient gene delivery into pituitary cells. Bromocriptine, a dopaminergic agonist known to decrease intracellular cAMP concentrations, caused inhibition of PRL promoter activity and lactotroph proliferation, which was accompanied by decreases in CRE-mediated transcription and CREB phosphorylation in lactotrophs. Expression of a dominant-negative form of CREB (MCREB), which was effective in suppressing CRE-mediated transcription induced by the adenylate cyclase activator forskolin, inhibited basal and forskolin-induced PRL promoter activity and PRL mRNA expression. MCREB expression lowered basal proliferative levels and blocked forskolin-induced proliferation of lactotrophs. Insulin-like growth factor I(IGF-I), a potent mitogen in lactotrophs, did not affect intracellular cAMP concentrations but transiently increased lactotroph CREB phosphorylation. MCREB expression also inhibited IGF-I-induced lactotroph proliferation. These results suggest that CREB is involved in the regulation of cell proliferation and the PRL promoter in normal lactotrophs and that dopamine inhibition of these lactotroph functions is at least in part due to inhibition of the cAMP-PKA-CREB pathway. adenosine 3',5'-cyclic monophosphate; dopamine; insulin-like growth factor I

In this report we describe a mathematical model for the regulation of cAMP dynamics in pancreatic [beta]-cells. Incretin hormones such as glucagon-like peptide 1 (GLP-1) increase cAMP and augment insulin secretion in pancreatic [beta]-cells. Imaging experiments performed in MIN6 insulinoma cells expressing a genetically encoded cAMP biosensor and loaded with fura-2, a calcium indicator, showed that cAMP oscillations are differentially regulated by periodic changes in membrane potential and GLP-1. We modeled the interplay of intracellular calcium ([Ca.sup.2+]) and its interaction with calmodulin, G protein-coupled receptor activation, adenylyl cyclases (AC), and phosphodiesterases (PDE). Simulations with the model demonstrate that cAMP oscillations are coupled to cytoplasmic [Ca.sup.2+] oscillations in the [beta]-cell. Slow [Ca.sup.2+] oscillations (3-4 [min.sup.-1]) entrain high-frequency, low-amplitude cAMP oscillations. The model predicts that GLP-1 receptor agonists induce cAMP oscillations in phase with cytoplasmic [Ca.sup.2+] oscillations. In contrast, observed antiphasic [Ca.sup.2+] and cAMP oscillations can be simulated following combined glucose and tetraethylammonium-induced changes in membrane potential. The model provides additional evidence for a pivotal role for [Ca.sup.2+]-dependent AC and PDE activation in coupling of [Ca.sup.2+] and cAMP signals. Our results reveal important differences in the effects of glucose/ TEA and GLP-1 on cAMP dynamics in MIN6 [beta]-cells. adenylyl cyclase; calcium ion; glucagon-like peptide 1; modeling; oscillations

Malignant gliomas are one of the leading causes of cancer deaths worldwide, but chemoprevention strategies for them are few and poorly investigated. Here, we show that cholera toxin, the traditional biotoxin and well known inducer of accumulation of cellular cAMP, is capable of inducing differentiation on malignant gliomas in vitro with rat C6 and primary cultured human glioma cells. Cholera toxin-induced differentiation was characterized by typical morphological changes, increased expression of glial fibrillary acid protein, decreased expression of Ki-67, inhibition of cellular proliferation, and accumulation of cells in the G1 phase of the cell cycle. Cholera toxin also triggered a significant reduction in the G1 cell-cycle regulatory proteins cyclin D1 and Cdk2 along with an overexpression of cell-cycle inhibitory proteins [p21.sup.Cip1] and [p27.sup.Kip1]. Abrogation of cAMP-dependent protein kinase A activity by protein kinase A inhibitor or silencing of cAMP-responsive element binding proteins by RNA interference resulted in suppressed differentiation. These findings imply the attractiveness of cholera toxin as a drug candidate for further development of differentiation therapy. Furthermore, activation of the protein kinase A/cAMP-responsive element binding protein pathway may be a key and requisite factor in glioma differentiation.

NaCl absorption by the thick ascending limb of Henle's loop (TAL) is mediated by the apical Na-K-2Cl cotransporter NKCC2. cAMP increases NaCl absorption in the TAL by stimulating NKCC2. In oocytes, cAMP increases NKCC2 activity by regulating its trafficking. However, the mechanism by which cAMP stimulates NKCC2 in TALs is not clear. We hypothesized that cAMP increases surface expression of NKCC2 and NaCl absorption in TALs and that vesicle-associated membrane protein (VAMP) is involved in this mechanism. We used surface biotinylation of rat medullary TALs (mTAL) to examine surface and total NKCC2 levels. When mTAL suspensions were treated with dibutyryl cAMP (db-cAMP) or forskolin plus IBMX for 20 min, surface NKCC2 expression increased by 126 [+ or -] 23 and 92 [+ or -] 17% above basal, respectively (P < 0.03). No changes in total NKCC2 expression were observed, suggesting that cAMP increased translocation of NKCC2. We studied the role of VAMP in NKCC2 translocation and found that incubating mTALs with tetanus toxin (30 nM), which inhibits vesicle trafficking by inactivating VAMP-2 and -3, completely blocked the stimulatory effect of db-cAMP on surface NKCC2 expression (tetanus toxin = 100% vs. tetanus toxin + db-cAMP = 102 [+ or -] 21% of control; not significant). We studied VAMP-2 and -3 expression and localization in isolated perfused TALs by confocal microscopy and found that both of them were located in the subapical space of the TAL. Finally, in isolated perfused mTALs, db-cAMP increased net Cl absorption by 95.0 [+ or -] 34.8% (P < 0.03), and pretreatment of TALs with tetanus toxin blocked the stimulation of Cl absorption (from 110.9 [+ or -] 15.9 to 109.7 [+ or -] 15.6 pmol x [min.sup.-1] x [mm.sup.-1]; not significant). We concluded that cAMP increases NKCC2 surface expression by a mechanism involving VAMP and that NKCC2 trafficking to the apical membrane is involved in the stimulation of TAL NaCl absorption by cAMP. Na-K-2Cl cotransporter; trafficking; vesicle-associated membrane protein

The ion transport defects reported for human cystic fibrosis (CF) airways are reproduced in nasal epithelia of the CF mouse. Although this tissue has been studied in vivo using the nasal potential difference technique and as a native tissue mounted in the Ussing chamber, little information is available on cultured murine nasal epithelia. We have developed a polarized cell culture model of primary murine nasal epithelia in which the CF tissue exhibits not only a defect in cAMP-ediated [Cl.sup.-] secretion but also the [Na.sup.+] hyperabsorption and upregulation of the [Ca.sup.2+]-activated [Cl.sup.-] conductance observed in human airways. Both the wild-type and CF cultures were constituted predominantly of undifferentiated cuboidal columnar cells, with most cultures exhibiting a small number of ciliated cells. Although no goblet cells were observed, RT-PCR demonstrated the expression of Muc5ac RNA after ~22 days in culture. The CF tissue exhibited an adherent layer of mucus similar to the mucus plaques reported in the distal airways of human CF patients. Furthermore, we found that treatment of CF preparations with a [Na.sup.+] channel blocker for 7 days prevented formation of mucus adherent to epithelial surfaces. The cultured murine nasal epithelial preparation should be an excellent model tissue for gene transfer studies and pharmacological studies of [Na.sup.+] channel blockers and mucolytic agents as well as for further characterization of CF ion transport defects. Culture of nasal epithelia from AF508 mice will be particularly useful in testing drugs that allow [DELTA]F508 CFTR to traffic to the membrane. [DELTA]F508 mice; cystic transmembrane conductance regulator; epithelial sodium channel; mucus; [Na.sup.+] hyperabsorption

Byline: E. V. Bigdai (1), V. O. Samoilov (1) Keywords: olfactory transduction; cyclic adenosine monophosphate system; fluorescent microscopy Abstract: Experiments on isolated frog olfactory epithelium, using vital luminescent microscopy showed that the olfactory transduction of amyl alcohol is mediated by the intracellular cAMP signaling system. Increases in intracellular cAMP levels resulted from activation of adenylate cyclase type III via odorant-induced stimulation of G protein linked to it. Author Affiliation: (1) I. P. Pavlov Institute of Physiology, Russian Academy of Sciences, 6 Makarov Bank, 199034, St. Petersburg, Russia Article History: Registration Date: 12/10/2004

Changes in hippocampal function seem critical for cognitive impairment in Alzheimer's disease (AD). Although there is eventual loss of synapses in both AD and animal models of AD, deficits in spatial memory and inhibition of long-term potentiation (LTP) precede morphological alterations in the models, suggesting earlier biochemical changes in the disease. In the studies reported here we demonstrate that amyloid [beta]-peptide (A[beta]) treatment of cultured hippocampal neurons leads to the inactivation of protein kinase A (PKA) and persistence of its regulatory subunit PKAII[alpha]. Consistent with this, CREB phosphorylation in response to glutamate is decreased, and the decrease is reversed by rolipram, a phosphodiesterase inhibitor that raises cAMP and leads to the dissociation of the PKA catalytic and regulatory subunits. It is likely that a similar mechanism underlies A[beta] inhibition of LTP, because rolipram and forskolin, agents that enhance the cAMP-signaling pathway, can reverse this inhibition. This reversal is blocked by H89, an inhibitor of PKA. These observations suggest that A[beta] acts directly on the pathways involved in the formation of late LTP and agents that enhance the cAMP/PKA/CREB-signaling pathway have potential for the treatment of AD.

Elevation of cAMP inhibits proliferation and expression of the transformed phenotype in several cell types. We studied the effects of elevation of cAMP and expression of mutant (Q227L) activated G[[alpha].sub.s] on the proliferation and tumorigenic capability of the later stage, metastatic estrogen-independent human breast cancer cell lines MDA-231 and MDA-435. Our studies show that 8Br-cAMP inhibits proliferation of these cells in culture and their ability to form colonies in soft agar. This inhibition may occur by different mechanisms in the two cell types. In MDA-231 cells, cAMP elevation results in sustained expression of the cell cycle inhibitor p27kip1 and inhibition of CDK2 activity, whereas in MDA-435 cells inhibition of mitogen-activated protein (MAP) kinase 1,2 activity is observed. We tested whether these effects in culture could be translated into inhibition of tumor growth in vivo. Tumors were developed in athymic (Nu/Nu) mice by injections of MDA-231 or MDA-435 cells. Injection of Q227L-G[[alpha].sub.s] expressing adenoviral vector into these established tumors inhibited further tumor growth under conditions where tumors injected with either saline or adenoviral vector containing [beta]-galactosidase grew up to four to five times their original size. These results raise the possibility that sustained elevation of cAMP may have therapeutic value in the treatment of estrogen-resistant later stage breast cancers.