Activation of cAMP-guanine exchange factor confers PKA-independent protection from hepatocyte apoptosis

cAMP has previously been shown to promote cell survival in a variety of cell types, but the downstream signaling pathway(s) of this antiapoptotic effect is unclear. Thus the role of cAMP signaling through PKA and cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs) in cAMP's antiapoptotic action was investigated in the present study. cAMP's protective effect against bile acid-, Fas ligand-, and TNF-alpha-induced apoptosis in rat hepatocytes was largely unaffected by the selective PKA inhibitor, Rp-8-(4-chlorophenylthio)-cAMP (Rp-cAMP). In contrast, a novel cAMP analog, 8-(4-chlorophenylthio)-2'-O-methyl (CPT-2-Me)-cAMP, which activated cAMP-GEFs in hepatocytes without activating PKA, protected hepatocytes against apoptosis induced by bile acids, Fas ligand, and TNF-alpha. The role of cAMP-GEF and PKA on activation of Akt, a kinase implicated in cAMP survival signaling, was investigated. Inhibition of PKA with RP-cAMP had no effect on cAMP-mediated Akt phosphorylation, whereas CPT-2-Me-cAMP, which did not activate PKA, induced phosphatidylinositol 3-kinase (PI3-kinase)-dependent activation of Akt. Pretreatment of hepatocytes with the PI3-kinase inhibitor, Ly-294002, prevented CPT-2-Me-cAMP's protective effect against bile acid and Fas ligand, but not TNF-alpha-mediated apoptosis. Glucagon, CPT-cAMP, and CPT-2-Me-cAMP all activated Rap 1, a downstream effector of cAMP-GEF. These results suggest that a PKA-independent cAMP/cAMP-GEF/Rap pathway exists in hepatocytes and that activation of cAMP-GEFs promotes Akt phosphorylation and hepatocyte survival. Thus a cAMP/cAMP-GEF/Rap/PI3-kinase/Akt signaling pathway may confer protection against bile acid- and Fas-induced apoptosis in hepatocytes.     

Parathyroid hormone-related protein regulates apoptosis in lung cancer cells through protein kinase A

Parathyroid hormone-related protein (PTHrP)-(1–34) and PTHrP-(140–173) protect lung cancer cells from apoptosis after ultraviolet (UV) irradiation. This study evaluated upstream signaling in PTHrP-mediated alteration of lung cancer cell sensitivity to apoptosis. The two peptides increased cAMP levels in BEN lung cancer cells by 15–35% in a dose-dependent fashion, suggesting signaling through protein kinase A (PKA). In line with this view, the PKA inhibitor H89 abrogated the protective effects of PTHrP-(1–34) and PTHrP-(140–173) against caspase activation and DNA loss. PKA activation by forskolin, 3-isobutyl-1-methylxanthine (IBMX), or 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate attenuated and H89 augmented apoptosis after UV exposure as indicated by caspase-3 activation, cell DNA loss, and morphological criteria. Studies with IBMX and varying doses of forskolin indicated that small increases in cAMP, on the order of those generated by IBMX alone and the PTHrP peptides, were sufficient to protect lung cancer cells from apoptosis. In summary, PTHrP-(1–34) and PTHrP-(140–173) stimulate PKA in lung carcinoma cells and protect cells against UV-induced caspase-3 activation and DNA fragmentation. PKA activation by other means also induces resistance to apoptosis, and the protective effect of the PTHrP peptide is blocked by PKA inhibition. Thus PKA appears to have a role in the regulatory effects of PTHrP on lung cancer cell survival. caspases; cell surface receptors; growth substances; signal transduction     

The biphasic induction of p21 and p27 in breast cancer cells by modulators of cAMP is posttranscriptionally regulated and independent of the PKA pathway.

Cyclic AMP (cAMP) elevation affects growth arrest and differentiation in a wide variety of breast cell lines; however, the mechanisms associated with this process are poorly understood. Previous studies linked cAMP-mediated growth arrest in breast tumor cells to increased levels of cyclin kinase inhibitor (CKI), p21. In the present study we examined the role of cAMP-dependent protein kinase (PKA) on p21 and p27 induction in the breast cancer cell line, MDA-MB-157. The induction of the CKIs by modulators of cAMP such as cholera toxin (CT) + 1-isobutyl-3-methylxanthine (IBMX) and lovastatin fluctuates with biphasic kinetics (although the kinetics of CKI induction with CT + IBMX treatment are different from that of lovastatin) and is depicted by the periodic accumulation of lower molecular weight forms of p21 and p27 which also correlate with fluctuations in CDK2 activity. Using three different approaches we show that the cAMP-mediated induction of CKIs is independent of PKA activity. In the first approach we treated MDA-MB-157 cells with a variety of cAMP modulators such as CT + IBMX, and forskolin in the presence or absence of H-89, a potent PKA inhibitor. This analysis revealed that the cAMP activators were capable of inducing p21 even though PKA activity was completely eliminated. In the second approach PKA dominant negative stable clones of MDA-MB-157 treated with CT + IBMX or forskolin also resulted in p21 induction, in the absence of any PKA activity. Last, treatment of MDA-MB-157 cells with lovastatin, another known cAMP modulator which also causes growth arrest, resulted in the induction of p21 and p27 without any increase in PKA activity. Collectively, the above results suggest that the induction of p21 by cAMP is through a novel pathway, independent of PKA activity.     

The cyclic AMP-protein kinase A pathway restrains islet phospholipase A(2) activation

Although phospholipase A(2) (PLA(2)) is of importance for insulin secretion, it is not established how it relates to other signalling mechanisms. This study examined the crosstalk between PLA(2) and the cyclic AMP (cAMP)-protein kinase A (PKA) pathway in isolated rat islets. Forskolin, IBMX, and dbcAMP reduced [(3)H]arachidonic acid ([(3)H]AA) efflux from prelabelled islets during PLA(2) activation by mellitin or cholecystokinin (CCK-8), while efflux induced by carbachol was unaffected. The PKA inhibitor myrPKI(14-22) prevented this reduction of CCK-8-induced efflux. Glucagon-like peptide-1 (GLP-1), gastric inhibitory polypeptide (GIP), and vasoactive intestinal polypeptide (VIP) diminished CCK-8-induced efflux. Also in the absence of Ca(2+), forskolin/IBMX and dbcAMP reduced CCK-8-induced efflux. In parallel with effects on [(3)H]AA, the expected additive insulin secretion induced by mellitin or CCK-8 in combination with forskolin or GLP-1, respectively, was reduced. In conclusion, the cAMP-PKA pathway restrains both Ca(2+)-dependent and Ca(2+)-independent PLA(2) activation, indicating a regulating crosstalk between these two pathways.     

Roles of protein kinase A and C in spontaneous maturation and in forskolin or 3-isobutyl-1-methylxanthine maintained meiotic arrest of bovine oocytes

Four hypotheses were tested using isolated bovine oocytes. (1) Cumulus oocyte complexes (COCs) or denuded oocytes (DOs) were cultured with the protein kinase A (PKA) inhibitor, H-89, to test if meiotic arrest induced by forskolin or IBMX was due to cAMP-stimulated PKA activity or nonspecific effects of these cAMP elevators. (2) COCs were cultured with a protein kinase C (PKC) stimulator (PDDβ) or inhibitor (GF109203x) to test if PKC modulation altered oocyte maturation. (3) COCs were prestimulated for 15 min with (a) PDDβ followed by cotreatment with forskolin, or (b) with H-89 or H-7 followed by cotreatment with GF109203x, to test for interaction between the PKA and PKC signal transduction pathways. (4) H-89 was added to spontaneously maturing COCs at intervals 0–18 hr to test if H-89 interfered with the transition between meiosis I and II. The results were as follows: H-89 interfered with forskolin or IBMX arrested oocytes in a dose-response manner (IBMX ED₅₀ = 41 μM for COCs; forskolin ED₅₀ = 9 μM for denuded oocytes). Prestimulation with PKC induced meiotic resumption in COCs in spite of the presence of forskolin [PDDβ followed by PDDβ + forskolin: 41–47% germinal vesicle (GV) oocytes; forskolin alone: 90–95% GV], while PKC inhibition induced meiotic arrest to a similar extent as forskolin (GF109230x, 85% GV; forskolin, 67–80% GV). Additionally, pretreatment of COCs with H-89 interfered with GF109203x induced arrest (41% vs. 90% GV, respectively). Finally, H-89 interfered with the timely progression of COCs from meiosis I and II. These results indicate that the PKA and PKC pathways can modulate the maturation of bovine oocytes in vitro. © 1996 Wiley-Liss, Inc.     

Extracellular ATP protects pancreatic duct epithelial cells from alcohol-induced damage through P2Y<Subscript>1</Subscript> receptor-cAMP signal pathway

Extracellular adenosine-5′-triphosphate (ATP) regulates cell death and survival of neighboring cells. The detailed effects are diverse depending on cell types and extracellular ATP concentration. We addressed the effect of ATP on ethanol-induced cytotoxicity in epithelial cells, the cell type that experiences the highest concentrations of alcohol. Using pancreatic duct epithelial cells (PDEC), we found that a micromolar range of ATP reverses all intracellular toxicity mechanisms triggered by exceptionally high doses of ethanol and, thus, improves cell viability dramatically. Out of the many purinergic receptors expressed in PDEC, the P2Y₁ receptor was identified to mediate the protective effect, based on pharmacological and siRNA assays. Activation of P2Y₁ receptors increased intracellular cyclic adenosine monophosphate (cAMP). The protective effect of ATP was mimicked by forskolin and 8-Br-cAMP but inhibited by a protein kinase A (PKA) inhibitor, H-89. Finally, ATP reverted leakiness of PDEC monolayers induced by ethanol and helped to maintain epithelial integrity. We suggest that purinergic receptors reduce extreme alcohol-induced cell damage via the cAMP signal pathway in PDEC and some other types of cells.     

Regulation of basal expression of catecholamine-synthesizing enzyme genes by PACAP.

We have previously reported that the cAMP/protein kinase A (PKA) pathway is important in the gene regulation of both induction and basal expressions of the catecholamine synthesizing enzymes tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) has been shown to activate the intracellular cAMP/PKA pathway. In the present study, using primary cultured bovine adrenal medullary cells, we determined whether the basal activity of the PACAP receptor might play a role in the maintenance of the basal expression of these enzyme genes via the cAMP/PKA pathway. The potent PACAP receptor antagonist PACAP (6-38) caused a reduction of TH and DBH mRNA levels in a dose dependent manner as well as their enzyme activities and TH protein level. The effects of PACAP (6-38) and the PKA inhibitor H-89 exhibited generally similar trends, and were not additive in the reduction of TH and DBH gene expression and activities, suggesting that they take a common intracellular signaling pathway. The antagonist also caused decreases in the intracellular norepinephrine and epinephrine levels similar to the effect of H-89. Taken together, the data suggests that PACAP is involved in the regulation of maintenance of the catecholamine synthesizing enzymes TH and DBH by utilizing the cAMP/PKA pathway.     

Systems analysis of GLP-1 receptor signaling in pancreatic β-cells

Glucagon-like peptide-1 (GLP-1) elevates intracellular concentration of cAMP ([cAMP]) and facilitates glucose-dependent insulin secretion in pancreatic β-cells. There has been much evidence to suggest that multiple key players such as the GLP-1 receptor, G(s) protein, adenylate cyclase (AC), phosphodiesterase (PDE), and intracellular Ca(2+) concentration ([Ca(2+)]) are involved in the regulation of [cAMP]. However, because of complex interactions among these signaling factors, the kinetics of the reaction cascade as well as the activities of ACs and PDEs have not been determined in pancreatic β-cells. We have constructed a minimal mathematical model of GLP-1 receptor signal transduction based on experimental findings obtained mostly in β-cells and insulinoma cell lines. By fitting this theoretical reaction scheme to key experimental records of the GLP-1 response, the parameters determining individual reaction steps were estimated. The model reconstructed satisfactorily the dynamic changes in [cAMP] and predicted the activities of cAMP effectors, protein kinase A (PKA), and cAMP-regulated guanine nucleotide exchange factor [cAMP-GEF or exchange protein directly activated by cAMP (Epac)] during GLP-1 stimulation. The simulations also predicted the presence of two sequential desensitization steps of the GLP1 receptor that occur with fast and very slow reaction rates. The cross talk between glucose- and GLP-1-dependent signal cascades for cAMP synthesis was well reconstructed by integrating the direct regulation of AC and PDE by [Ca(2+)]. To examine robustness of the signaling system in controlling [cAMP], magnitudes of AC and PDE activities were compared in the presence or absence of GLP-1 and/or the PDE inhibitor IBMX.(1).     

Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent inhibition of IL-5 from human T lymphocytes is not mediated by the cAMP-dependent protein kinase A

IL-5 is implicated in the pathogenesis of asthma and is predominantly released from T lymphocytes of the Th2 phenotype. In anti-CD3 plus anti-CD28-stimulated PBMC, albuterol, isoproterenol, rolipram, PGE2, forskolin, cholera toxin, and the cAMP analog, 8-bromoadenosine cAMP (8-Br-cAMP) all inhibited the release of IL-5 and lymphocyte proliferation. Although all of the above compounds share the ability to increase intracellular cAMP levels and activate protein kinase (PK) A, the PKA inhibitor H-89 failed to ablate the inhibition of IL-5 production mediated by 8-Br-cAMP, rolipram, forskolin, or PGE2. Similarly, H-89 had no effect on the cAMP-mediated inhibition of lymphocyte proliferation. Significantly, these observations occurred at a concentration of H-89 (3 microM) that inhibited both PKA activity and CREB phosphorylation in intact cells. Additional studies showed that the PKA inhibitors H-8, 8-(4-chlorophenylthio) adenosine-3',5'-cyclic monophosphorothioate Rp isomer, and a myristolated PKA inhibitor peptide also failed to block the 8-Br-cAMP-mediated inhibition of IL-5 release from PBMC. Likewise, a role for PKG was considered unlikely because both activators and inhibitors of this enzyme had no effect on IL-5 release. Western blotting identified Rap1, a downstream target of the cAMP-binding proteins, exchange protein directly activated by cAMP/cAMP-guanine nucleotide exchange factors 1 and 2, in PBMC. However, Rap1 activation assays revealed that this pathway is also unlikely to be involved in the cAMP-mediated inhibition of IL-5. Taken together, these results indicate that cAMP-elevating agents inhibit IL-5 release from PBMC by a novel cAMP-dependent mechanism that does not involve the activation of PKA.     

Modulation of dihydropyridine-sensitive calcium channels in Drosophila by a cAMP-mediated pathway.

Drosophila has proved to be a valuable system for studying the structure and function of ion channels. However, relatively little is known about the regulation of ion channels, particularly that of Ca2+ channels, in Drosophila. Physiological and pharmacological differences between invertebrate and mammalian L-type Ca2+ channels raise questions on the extent of conservation of Ca2+ channel modulatory pathways. We have examined the role of cyclic adenosine monophosphate (cAMP) cascade in modulating the dihydropyridine (DHP)-sensitive Ca2+ channels in the larval muscles of Drosophila, using mutations and drugs that disrupt specific steps in this pathway. The L-type (DHP-sensitive) Ca2+ channel current was increased in the dunce mutants, which have high cAMP concentration owing to cAMP-specific phosphodiesterase (PDE) disruption. The current was decreased in the rutabaga mutants, where adenylyl cyclase (AC) activity is altered thereby decreasing the cAMP concentration. The dunce effect was mimicked by 8-Br-cAMP, a cAMP analog, and IBMX, a PDE inhibitor. The rutabaga effect was rescued by forskolin, an AC activator. H-89, an inhibitor of protein kinase-A (PKA), reduced the current and inhibited the effect of 8-Br-cAMP. The data suggest modulation of L-type Ca2+ channels of Drosophila via a cAMP-PKA mediated pathway. While there are differences in L-type channels, as well as in components of cAMP cascade, between Drosophila and vertebrates, main features of the modulatory pathway have been conserved. The data also raise questions on the likely role of DHP-sensitive Ca2+ channel modulation in synaptic plasticity, and learning and memory, processes disrupted by the dnc and the rut mutations.     

Methoxyflavones protect cells against endoplasmic reticulum stress and neurotoxin

Enhanced endoplasmic reticulum (ER) stress leads to cell death in various pathophysiological situations. During a search for compounds that regulate ER stress, we identified methoxyflavones, a group of flavonoids, as strong protective agents against ER stress. Analysis in mouse insulinoma MIN6 cells revealed that methoxyflavones mildly activated the eukaryotic initiation factor 2 and nuclear factor erythroid 2-related factor pathways, but not the XBP1 pathway, and induced downstream genes, including glucose-regulated protein (GRP) 78, a molecular chaperone in the ER. The protective effect of methoxyflavones was enhanced by agents that increase intracellular cAMP levels such as forskolin, dibutyryl-cAMP and IBMX, but suppressed by the protein kinase A (PKA) inhibitor H-89, suggesting involvement of the PKA pathway in the regulation of ER stress by methoxyflavones. Consistent with the results in cultured cells, pretreatment of mice with tangeretin, a methoxyflavone, enhanced expression of GRP78 and HO-1 without causing ER stress in renal tubular epithelium and prevented tunicamycin-induced cell death. Furthermore, preadministration of tangeretin in mice enhanced expression of GRP78 in the substantia nigra pars compacta and protected dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a neurotoxin that induces both oxidative and ER stress. These results suggest that methoxyflavones play an important role in the regulation of ER stress and could be a therapeutic target for the ER stress-related diseases. neuronal degeneration; flavonoids     

Self-renewal and differentiation of mouse embryonic stem cells as measured by Oct4 expression: the role of the cAMP/PKA pathway

In this study we examined the role of the cAMP/protein kinase A (PKA) pathway in affecting IOUD2 ES cell self-renewal and differentiation, Oct4 expression, and cell proliferation. Forskolin, the adenylate cyclase agonist, alone had no effect on ES cell self-renewal. However, when cells were treated with the differentiation-inducing agent retinoic acid, forskolin significantly promoted ES cell self-renewal. Effectively, forskolin rescued cells from a pathway of differentiation. Culturing ES cells in the presence of the phosphodiesterase inhibitor IBMX had no effect on ES cell self-renewal but did increase cell proliferation. In the presence of 100 μM IBMX without LIF, 10 μM forskolin significantly increased ES cell self-renewal. The cell permeable cAMP analog 8-Br-cAMP (1 and 5 mM) promoted ES cell differentiation in the presence of LIF, while in the absence of LIF, it promoted ES cell self-renewal. The effect of the PKA specific inhibitors H89 and KT5720 on Oct4 expression was, again, LIF-dependent. In the presence of LIF, these inhibitors decreased Oct4 expression, while they increased Oct4 expression in the absence of LIF. In general, ES cells maintained on a self-renewal pathway through the presence of LIF show little effect from altered cAMP signaling except at higher levels. However, in strict contrast, when ES cell are on a differentiation pathway through exposure to retinoic acid or the removal of LIF, altering cAMP levels can rescue the self-renewal process promoting Oct4 expression. This study clearly shows that the cAMP/PKA pathway plays a role in ES cell self-renewal pathways. This work was partly funded by the Millennium Research Fund National University of Ireland Galway.     

cAMP-dependent protein kinase inhibits the mitogenic action of vascular endothelial growth factor and fibroblast growth factor in capillary endothelial cells by blocking Raf activation

Proliferation of endothelial cells is regulated by angiogenic and antiangiogenic factors whose actions are mediated by complex interactions of multiple signaling pathways. Both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) stimulate cell proliferation and activate the mitogen-activated protein kinase (MAPK) cascade in bovine brain capillary endothelial (BBE) cells. We have extended these findings to show that both mitogens activate MAPK via stimulation of Raf-1. Activation of Raf/MAPK is inhibited by increasing intracellular cAMP levels pharmacologically or via stimulation of endogenously expressed β-adrenergic receptors. Both VEGF- and bFGF-induced Raf-1 activity are blocked in the presence of forskolin or 8-bromo-cAMP by 80%. The actions of increased cAMP appear to be mediated by cAMP-dependent protein kinase (PKA), since treatment with H-89, a the specific inhibitor of PKA, reversed the inhibitory effect of elevated cAMP levels on mitogen-induced cell proliferation and Raf/MAPK activation. Moreover, elevations in cAMP/PKA activity inhibit mitogen-induced cell proliferation. These findings demonstrate, in cultured endothelial cells, that the cAMP/PKA signaling pathway is potentially an important physiological inhibitor of mitogen activation of the MAPK cascade and cell proliferation. J. Cell. Biochem. 67:353–366, 1997. © 1997 Wiley-Liss, Inc.     

Cyclic AMP signaling stimulates proteasome degradation of thioredoxin interacting protein (TxNIP) in pancreatic β-cells

Thioredoxin interacting protein (TxNIP) functions as an effector of glucotoxicity in pancreatic β-cells. Exendin-4 (Ex-4), a long-term effective GLP-1 receptor agonist, reduces TxNIP level in pancreatic β-cells. Mechanisms underlying this reduction, however, remain largely unknown. We show here that Ex-4, 8-bromo-cAMP, the cAMP promoting agent forskolin, as well as activators of protein kinase A (PKA) and exchange protein activated by cAMP (Epac), all attenuated the effect of high glucose (20 mM) on TxNIP level in the pancreatic β-cell line Ins-1. Forskolin and Ex-4 also reduced TxNIP level in cultured primary rat islets. This repressive effect is at least partially mediated via stimulating proteasome-dependent TxNIP degradation, since the proteasomal inhibitor MG132, but not the lysosomal inhibitor chloroquine, significantly blocked the repressive effect of forskolin. Furthermore, forskolin enhanced TxNIP ubiquitination. Both PKA inhibition and Epac inhibition partially blocked the repressive effect of forskolin on TxNIP level. In addition, forskolin and Ex-4 protected Ins-1 cells from high glucose-induced apoptotic activity, assessed by measuring caspase 3 activity. Finally, knockdown of TxNIP expression led to reduced caspase 3 expression levels and blunted response to forskolin treatment. We suggest that proteasome-dependent TxNIP degradation is a novel mechanism by which Ex-4-cAMP signaling protects pancreatic β cells.     

Protein secretion in cockroach salivary glands requires an increase in intracellular cAMP and Ca2+ concentrations

The salivary glands in the cockroach Periplaneta americana secrete protein-containing saliva when stimulated by serotonin (5-HT) and protein-free saliva upon dopamine stimulation. In order to obtain information concerning the signalling pathways involved in 5-HT-induced protein secretion, we have determined the protein content of saliva secreted after experimental manipulations that potentially elevate intracellular Ca2+ and cyclic nucleotide concentrations in isolated glands. We have found that 5-HT stimulates the rate of protein secretion in a dose-dependent manner (threshold: 3 x 10(-8)M; EC50 1.5 x 10(-6)M). The maximal rate of 5-HT-induced protein secretion was 2.2 +/- 0.2 microg/min. Increasing intracellular Ca2+ or cAMP by bath application of ionomycin (5 microM), db cAMP (10mM), forskolin (100 microM) or IBMX (100 microM), respectively, stimulated protein secretion at significantly lower rates, whereas db cGMP (1mM) did not activate protein secretion. The high rates and the kinetics of 5-HT-induced protein secretion could only be mimicked by either applying forskolin together with IBMX (with or without ionomycin) or by applying IBMX together with ionomycin. Our measurements suggest that 5-HT-induced protein secretion is mediated by an elevation of [cAMP]i and that Ca2+ may function as a co-agonist and augment the rate of protein secretion.     

PDE4 inhibitor, roflumilast protects cardiomyocytes against NO-induced apoptosis via activation of PKA and Epac dual pathways

Myocyte apoptosis plays an important role in myocardial infarction and cAMP is crucial in the regulation of myocyte apoptosis. Phosphodiesterase-4 (PDE4) inhibitor blocks the hydrolysis of cAMP via inhibition of PDE4 and is attractive candidate for novel anti-inflammatory drugs. However, its function in cardiovascular diseases and cardiomyocyte apoptosis is unclear. Therefore, we investigated whether roflumilast, a PDE4 inhibitor, exerts protective effect against NO-induced apoptosis in both of H9c2 cells and neonatal rat cardiomyocytes (NRCMs), focusing on cAMP downstream molecules such as protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). According to our data, intracellular cAMP was increased by roflumilast treatment in H9c2 cells and NRCMs. Roflumilast inhibited SNP-induced apoptosis and this effect was reversed by PKA specific inhibitor H-89 and KT-5720. In addition, PKA specific activator N(6)-benzoyladenosine 3',5-cyclic monophosphate (N(6)Bz-cAMP) mimicked the effects of roflumilast. CREB phosphorylation by roflumilast was also inhibited by H-89, indicating that roflumilast protects SNP-induced apoptosis via PKA-dependent pathway. Roflumilast increased Epac1/GTP-Rap1 and the protective effect was abolished by Epac1 siRNA transfection, demonstrating that Epac signaling was also involved in this protective response. In support, Epac specific activator 8-(4-chlrorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8CPT-2Me-cAMP) protected SNP-induced apoptosis. PI3K/Akt inhibitor LY294002 blocked roflumilast-induced Akt phosphorylation and protective effect. Furthermore, inhibition of Epac1 with siRNA had no effect on roflumilast-induced CREB phosphorylation, whereas inhibited Akt phosphorylation, implicating that Akt phosphorylation was regulated by Epac pathway. In addition, it was also observed that rolipram and cilomilast exert similar effects as roflumilast. In summary, our data indicate that roflumilast protects NO-induced apoptosis via both cAMP-PKA/CREB and Epac/Akt-dependent pathway. Our study suggests a possibility of PDE4 inhibitor roflumilast as a potential therapeutic agent against myocardial ischemia/reperfusion (I/R) injury.     

Effect of GLP-1 on lipid metabolism in human adipocytes.

We have studied the effect of several doses of GLP-1, compared to that of insulin and glucagons, on lipogenesis, lipolysis and cAMP cellular content, in human adipocytes isolated from normal subjects. In human adipocytes, GLP-1 exerts a dual action, depending upon the dose, on lipid metabolism, being lipogenic at low concentrations of the peptide (ED50, 10(-12) M), and lipolytic only at doses 10-100 times higher (ED50, 10(-10) M); both effects are time- and GLP-1 concentration-dependent. The GLP-1 lipogenic effect is equal in magnitude to that of equimolar amounts of insulin; both hormones apparently act synergically, and their respective action is abolished by glucagon. The lipolytic effect of GLP-1 is comparable to that of glucagon, apparently additive to it, and the stimulated value induced by either one is neutralized by the presence of insulin. In the absence of IBMX, GLP-1, at 10(-13) and 10(-12) M, only lipogenic doses, does not modify the cellular content of cAMP, while from 10(-11) M to 10(-9) M, also lipolytic concentrations, it has an increasing effect; in the presence of IBMX, GLP-1 at already 10(-12) M increased the cellular cAMP content. In human adipocytes, GLP-1 shows glucagon- and also insulin-like effects on lipid metabolism, suggesting the possibility of GLP-1 activating two distinct receptors, one of them similar or equal to the pancreatic one, accounting cAMP as a second messenger only for the lipolytic action of the peptide.