Live Cell Monitoring of Phosphodiesterase Inhibition by Sulfonylurea Drugs.

Sulfonylureas (SUs) are a class of antidiabetic drugs widely used in the management of diabetes mellitus type 2. They promote insulin secretion by inhibiting the ATP-sensitive potassium channel in pancreatic ß-cells. Recently, the exchange protein directly activated by cAMP (Epac) was identified as a new class of target proteins of SUs that might contribute to their antidiabetic effect, through the activation of the Ras-like guanosine triphosphatase Rap1, which has been controversially discussed. We used human embryonic kidney (HEK) 293 cells expressing genetic constructs of various Förster resonance energy transfer (FRET)-based biosensors containing different versions of Epac1 and Epac2 isoforms, alone or fused to different phosphodiesterases (PDEs), to monitor SU-induced conformational changes in Epac or direct PDE inhibition in real time. We show that SUs can both induce conformational changes in the Epac2 protein but not in Epac1, and directly inhibit the PDE3 and PDE4 families, thereby increasing cAMP levels in the direct vicinity of these PDEs. Furthermore, we demonstrate that the binding site of SUs in Epac2 is distinct from that of cAMP and is located between the amino acids E443 and E460. Using biochemical assays, we could also show that tolbutamide can inhibit PDE activity through an allosteric mechanism. Therefore, the cAMP-elevating capacity due to allosteric PDE inhibition in addition to direct Epac activation may contribute to the therapeutic effects of SU drugs.

cAMP-Epac1 signaling is activated in DDAVP-induced endolymphatic hydrops of guinea pigs

Abstract Objective To explore whether Cyclic Adenosine Monophosphate (cAMP)-Epac1 signaling is activated in 1-Desamino-8-D-arginine-Vasopressin-induced Endolymphatic Hydrops (DDAVP-induced EH) and to provide new insight for further in-depth study of DDAVP-induced EH. Methods Eighteen healthy, red-eyed guinea pigs (36 ears) weighing 200-350 g were randomly divided into three groups: the control group, which received intraperitoneal injection of sterile saline (same volume as that in the other two groups) for 7 consecutive days; the DDAVP-7d group, which received intraperitoneal injection of 10 mg/mL/kg DDAVP for 7 consecutive days; and the DDAVP-14d group, which received intraperitoneal injection of 10 μg/mL/kg DDAVP for 14 consecutive days. After successful modeling, all animals were sacrificed, and cochlea tissues were collected to detect the mRNA and protein expression of the exchange protein directly activated by cAMP-1 and 2 (Epac1, Epac2), and Repressor Activator Protein-1 (Rap1) by Reverse Transcription (RT)-PCR and western blotting, respectively. Results Compared to the control group, the relative mRNA expression of Epac1, Epac2, Rap1A, and Rap1B in the cochlea tissue of the DDAVP-7d group was significantly higher (p< 0.05), while no significant difference in Rap1 GTPase activating protein (Rap1gap) mRNA expression was found between the two groups. The relative mRNA expression of Epac1, Rap1A, Rap1B, and Rap1gap in the cochlea tissue of the DDAVP-14d group was significantly higher than that of the control group (p< 0.05), while no significant difference in Epac2 mRNA expression was found between the DDAVP-14d and control groups. Comparison between the DDAVP-14d and DDAVP-7d groups showed that the DDAVP-14d group had significantly lower Epac2 and Rap1A (p< 0.05) and higher Rap1gap (p < 0.05) mRNA expression in the cochlea tissue than that of the DDAVP-7d group, while no significant differences in Epac1 and Rap1B mRNA expression were found between the two groups. Western blotting showed that Epac1 protein expression in the cochlea tissue was the highest in the DDAVP-14d group, followed by that in the DDAVP-7d group, and was the lowest in the control group, showing significant differences between groups (p< 0.05); Rap1 protein expression in the cochlea tissue was the highest in the DDAVP-7d group, followed by the DDAVP-14d group, and was the lowest in the control group, showing significant differences between groups (p< 0.05); no significant differences in Epac2 protein expression in the cochlea tissue were found among the three groups. Conclusion DDAVP upregulated Epac1 protein expression in the guinea pig cochlea, leading to activation of the inner ear cAMP-Epac1 signaling pathway. This may be an important mechanism by which DDAVP regulates endolymphatic metabolism to induce EH and affect inner ear function. Oxford Centre for Evidence-Based Medicine 2011 Levels of Evidence Level 5.

cAMP-Epac1 signaling is activated in DDAVP-induced endolymphatic hydrops of guinea pigs.

OBJECTIVE: To explore whether Cyclic Adenosine Monophosphate (cAMP)-Epac1 signaling is activated in 1-Desamino-8-D-arginine-Vasopressin-induced Endolymphatic Hydrops (DDAVP-induced EH) and to provide new insight for further in-depth study of DDAVP-induced EH. METHODS: Eighteen healthy, red-eyed guinea pigs (36 ears) weighing 200-350 g were randomly divided into three groups: the control group, which received intraperitoneal injection of sterile saline (same volume as that in the other two groups) for 7 consecutive days; the DDAVP-7d group, which received intraperitoneal injection of 10 mg/mL/kg DDAVP for 7 consecutive days; and the DDAVP-14d group, which received intraperitoneal injection of 10 µg/mL/kg DDAVP for 14 consecutive days. After successful modeling, all animals were sacrificed, and cochlea tissues were collected to detect the mRNA and protein expression of the exchange protein directly activated by cAMP-1 and 2 (Epac1, Epac2), and Repressor Activator Protein-1 (Rap1) by Reverse Transcription (RT)-PCR and western blotting, respectively. RESULTS: Compared to the control group, the relative mRNA expression of Epac1, Epac2, Rap1A, and Rap1B in the cochlea tissue of the DDAVP-7d group was significantly higher (p <  0.05), while no significant difference in Rap1 GTPase activating protein (Rap1gap) mRNA expression was found between the two groups. The relative mRNA expression of Epac1, Rap1A, Rap1B, and Rap1gap in the cochlea tissue of the DDAVP-14d group was significantly higher than that of the control group (p <  0.05), while no significant difference in Epac2 mRNA expression was found between the DDAVP-14d and control groups. Comparison between the DDAVP-14d and DDAVP-7d groups showed that the DDAVP-14d group had significantly lower Epac2 and Rap1A (p <  0.05) and higher Rap1gap (p < 0.05) mRNA expression in the cochlea tissue than that of the DDAVP-7d group, while no significant differences in Epac1 and Rap1B mRNA expression were found between the two groups. Western blotting showed that Epac1 protein expression in the cochlea tissue was the highest in the DDAVP-14d group, followed by that in the DDAVP-7d group, and was the lowest in the control group, showing significant differences between groups (p <  0.05); Rap1 protein expression in the cochlea tissue was the highest in the DDAVP-7d group, followed by the DDAVP-14d group, and was the lowest in the control group, showing significant differences between groups (p <  0.05); no significant differences in Epac2 protein expression in the cochlea tissue were found among the three groups. CONCLUSION: DDAVP upregulated Epac1 protein expression in the guinea pig cochlea, leading to activation of the inner ear cAMP-Epac1 signaling pathway. This may be an important mechanism by which DDAVP regulates endolymphatic metabolism to induce EH and affect inner ear function. OXFORD CENTRE FOR EVIDENCE-BASED MEDICINE 2011 LEVELS OF EVIDENCE: Level 5.

Differential changes in cyclic adenosine 3'-5' monophosphate (cAMP) effectors and major Ca2+ handling proteins during diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM) is associated with differential and time-specific regulation of ß-adrenergic receptors and cardiac cyclic nucleotide phosphodiesterases with consequences for total cyclic adenosine 3'-5' monophosphate (cAMP) levels. We aimed to investigate whether these changes are associated with downstream impairments in cAMP and Ca2+ signalling in a type 1 diabetes (T1D)-induced DCM model. T1D was induced in adult male rats by streptozotocin (65 mg/kg) injection. DCM was assessed by cardiac structural and molecular remodelling. We delineated sequential changes affecting the exchange protein (Epac1/2), cAMP-dependent protein kinase A (PKA) and Ca2+ /Calmodulin-dependent kinase II (CaMKII) at 4, 8 and 12 weeks following diabetes, by real-time quantitative PCR and western blot. Expression of Ca2+ ATPase pump (SERCA2a), phospholamban (PLB) and Troponin I (TnI) was also examined. Early upregulation of Epac1 transcripts was noted in diabetic hearts at Week 4, followed by increases in Epac2 mRNA, but not protein levels, at Week 12. Expression of PKA subunits (RI, RIIα and Cα) remained unchanged regardless of the disease stage, whereas CaMKII increased at Week 12 in DCM. Moreover, PLB transcripts were upregulated in diabetic hearts, whereas SERCA2a and TnI gene expression was unchanged irrespective of the disease evolution. PLB phosphorylation at threonine-17 was increased in DCM, whereas phosphorylation of both PLB at serine-16 and TnI at serine-23/24 was unchanged. We show for the first time differential and time-specific regulations in cardiac cAMP effectors and Ca2+ handling proteins, data that may prove useful in proposing new therapeutic approaches in T1D-induced DCM.

Soluble adenylyl cyclase regulates the cytosolic NADH/NAD+ redox state and the bioenergetic switch between glycolysis and oxidative phosphorylation.

The evolutionarily conserved soluble adenylyl cyclase (sAC, ADCY10) mediates cAMP signaling exclusively in intracellular compartments. Because sAC activity is sensitive to local concentrations of ATP, bicarbonate, and free Ca2+, sAC is potentially an important metabolic sensor. Nonetheless, little is known about how sAC regulates energy metabolism in intact cells. In this study, we demonstrated that both pharmacological and genetic suppression of sAC resulted in increased lactate secretion and decreased pyruvate secretion in multiple cell lines and primary cultures of mouse hepatocytes and cholangiocytes. The increased extracellular lactate-to-pyruvate ratio upon sAC suppression reflected an increased cytosolic free [NADH]/[NAD+] ratio, which was corroborated by using the NADH/NAD+ redox biosensor Peredox-mCherry. Mechanistic studies in permeabilized HepG2 cells showed that sAC inhibition specifically suppressed complex I of the mitochondrial respiratory chain. A survey of cAMP effectors revealed that only selective inhibition of exchange protein activated by cAMP 1 (Epac1), but not protein kinase A (PKA) or Epac2, suppressed complex I-dependent respiration and significantly increased the cytosolic NADH/NAD+ redox state. Analysis of the ATP production rate and the adenylate energy charge showed that inhibiting sAC reciprocally affects ATP production by glycolysis and oxidative phosphorylation while maintaining cellular energy homeostasis. In conclusion, our study shows that, via the regulation of complex I-dependent mitochondrial respiration, sAC-Epac1 signaling regulates the cytosolic NADH/NAD+ redox state, and coordinates oxidative phosphorylation and glycolysis to maintain cellular energy homeostasis. As such, sAC is effectively a bioenergetic switch between aerobic glycolysis and oxidative phosphorylation at the post-translational level.

Exchange protein directly activated by cAMP (Epac) 1 plays an essential role in stress-induced exercise capacity by regulating PGC-1α and fatty acid metabolism in skeletal muscle.

Exchange protein directly activated by cAMP (Epac) mediates cAMP-mediated cell signal independent of protein kinase A (PKA). Mice lacking Epac1 displayed metabolic defect suggesting possible functional involvement of skeletal muscle and exercise capacity. Epac1 was highly expressed, but not Epac 2, in the extensor digitorum longus (EDL) and soleus muscles. The exercise significantly increased protein expression of Epac 1 in EDL and soleus muscle of wild-type (WT) mice. A global proteomics and pathway analyses revealed that Epac 1 deficiency mainly affected "the energy production and utilization" process in the skeletal muscle. We have tested their forced treadmill exercise tolerance. Epac1-/- mice exhibited significantly reduced exercise capacity in the forced treadmill exercise and lower number of type 1 fibers than WT mice. The basal protein level of proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) was reduced in the Epac1-/- mice. Furthermore, increasing expression of PGC-1α by exercise was also significantly attenuated in the skeletal muscle of Epac1-/- mice. The expressions of downstream target genes of PGC-1α, which involved in uptake and oxidation of fatty acids, ERRα and PPARδ, and fatty acid content were lower in muscles of Epac1-/-, suggesting a role of Epac1 in forced treadmill exercise capacity by regulating PGC-1α pathway and lipid metabolism in skeletal muscle. Taken together, Epac1 plays an important role in exercise capacity by regulating PGC-1α and fatty acid metabolism in the skeletal muscle.

Epac1 null mice have nephrogenic diabetes insipidus with deficient corticopapillary osmotic gradient and weaker collecting duct tight junctions.

AIM: The cAMP-mediator Epac1 (RapGef3) has high renal expression. Preliminary observations revealed increased diuresis in Epac1-/- mice. We hypothesized that Epac1 could restrict diuresis by promoting transcellular collecting duct (CD) water and urea transport or by stabilizing CD paracellular junctions to reduce osmolyte loss from the renal papillary interstitium. METHODS: In Epac1-/- and Wt C57BL/6J mice, renal papillae, dissected from snap-frozen kidneys, were assayed for the content of key osmolytes. Cell junctions were analysed by transmission electron microscopy. Urea transport integrity was evaluated by urea loading with 40% protein diet, endogenous vasopressin production was manipulated by intragastric water loading and moderate dehydration and vasopressin type 2 receptors were stimulated selectively by i.p.-injected desmopressin (dDAVP). Glomerular filtration rate (GFR) was estimated as [14 C]inulin clearance. The glomerular filtration barrier was evaluated by urinary albumin excretion and microvascular leakage by the renal content of time-spaced intravenously injected 125 I- and 131 I-labelled albumin. RESULTS: Epac1-/- mice had increased diuresis and increased free water clearance under antidiuretic conditions. They had shorter and less dense CD tight junction (TJs) and attenuated corticomedullary osmotic gradient. Epac1-/- mice had no increased protein diet-induced urea-dependent osmotic diuresis, and expressed Wt levels of aquaporin-2 (AQP-2) and urea transporter A1/3 (UT-A1/3). Epac1-/- mice had no urinary albumin leakage and unaltered renal microvascular albumin extravasation. Their GFR was moderately increased, unless when treated with furosemide. CONCLUSION: Our results conform to the hypothesis that Epac1-dependent mechanisms protect against diabetes insipidus by maintaining renal papillary osmolarity and the integrity of CD TJs.

Cyclic-AMP induces Nogo-A receptor NgR1 internalization and inhibits Nogo-A-mediated collapse of growth cone.

The promotion of axonal regeneration is required for functional recovery from stroke and various neuronal injuries. However, axonal regeneration is inhibited by diverse axonal growth inhibitors, such as Nogo-A. Nogo-66, a C-terminal domain of Nogo-A, binds to the Nogo-A receptor 1 (NgR1) and induces the collapse of growth cones and inhibits neurite outgrowth. NgR1 is also a receptor for additional axonal growth inhibitors, suggesting it is an important target for the prevention of axonal growth inhibition. By using the indirect immunofluorescence method, we show for the first time that a cell-permeable cAMP analog (dibutyryl-cAMP) induced a rapid decrease in the cell surface expression of NgR1 in Neuroscreen-1 (NS-1) cells. The biotinylation method revealed that cAMP indeed induced internalization of NgR1 within minutes. Other intracellular cAMP-elevating agents, such as forskolin, which directly activates adenylyl cyclase, and rolipram, which inhibits cyclic nucleotide phosphodiesterase, also induced this process. This internalization was found to be reversible and influenced by intracellular levels of cAMP. Using selective activators and inhibitors of protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac), we found that NgR1 internalization is independent of PKA, but dependent on Epac. The decrease in cell surface expression of NgR1 desensitized NS-1 cells to Nogo-66-induced growth cone collapse. Therefore, it is likely that besides axonal growth inhibitors affecting neurons, neurons themselves also self-regulate their sensitivity to axonal growth inhibitors, as influenced by intracellular cAMP/Epac. This normal cellular regulatory mechanism may be pharmacologically exploited to overcome axonal growth inhibitors, and enhance functional recovery after stroke and neuronal injuries.

Correlative Study on Impaired Prostaglandin E2 Regulation in Epicardial Adipose Tissue and its Role in Maladaptive Cardiac Remodeling via EPAC2 and ST2 Signaling in Overweight Cardiovascular Disease Subjects.

There is recent evidence that the dysfunctional responses of a peculiar visceral fat deposit known as epicardial adipose tissue (EAT) can directly promote cardiac enlargement in the case of obesity. Here, we observed a newer molecular pattern associated with LV dysfunction mediated by prostaglandin E2 (PGE2) deregulation in EAT in a cardiovascular disease (CVD) population. A series of 33 overweight CVD males were enrolled and their EAT thickness, LV mass, and volumes were measured by echocardiography. Blood, plasma, EAT, and SAT biopsies were collected for molecular and proteomic assays. Our data show that PGE2 biosynthetic enzyme (PTGES-2) correlates with echocardiographic parameters of LV enlargement: LV diameters, LV end diastolic volume, and LV masses. Moreover, PTGES-2 is directly associated with EPAC2 gene (r = 0.70, p < 0.0001), known as a molecular inducer of ST2/IL-33 mediators involved in maladaptive heart remodelling. Furthermore, PGE2 receptor 3 (PTEGER3) results are downregulated and its expression is inversely associated with ST2/IL-33 expression. Contrarily, PGE2 receptor 4 (PTGER4) is upregulated in EAT and directly correlates with ST2 molecular expression. Our data suggest that excessive body fatness can shift the EAT transcriptome to a pro-tissue remodelling profile, may be driven by PGE2 deregulation, with consequent promotion of EPAC2 and ST2 signalling.

Exchange protein directly activated by cAMP (Epac) protects against airway inflammation and airway remodeling in asthmatic mice.

BACKGROUND: ß2 receptor agonists induce airway smooth muscle relaxation by increasing intracellular cAMP production. PKA is the traditional downstream signaling pathway of cAMP. Exchange protein directly activated by cAMP (Epac) was identified as another important signaling molecule of cAMP recently. The role of Epac in asthmatic airway inflammation and airway remodeling is unclear. METHODS: We established OVA-sensitized and -challenged acute and chronic asthma mice models to explore the expression of Epac at first. Then, airway inflammation and airway hyperresponsiveness in acute asthma mice model and airway remodeling in chronic asthma mice model were observed respectively after treatment with Epac-selective cAMP analogue 8-pCPT-2'-O-Me-cAMP (8pCPT) and Epac inhibitor ESI-09. Next, the effects of 8pCPT and ESI-09 on the proliferation and apoptosis of in vitro cultured mouse airway smooth muscle cells (ASMCs) were detected with CCK-8 assays and Annexin-V staining. Lastly, the effects of 8pCPT and ESI-09 on store-operated Ca2+ entry (SOCE) of ASMCs were examined by confocal Ca2+ fluorescence measurement. RESULTS: We found that in lung tissues of acute and chronic asthma mice models, both mRNA and protein expression of Epac1 and Epac2, two isoforms of Epac, were lower than that of control mice. In acute asthma mice model, the airway inflammatory cell infiltration, Th2 cytokines secretion and airway hyperresponsiveness were significantly attenuated by 8pCPT and aggravated by ESI-09. In chronic asthma mice model, 8pCPT decreased airway inflammatory cell infiltration and airway remodeling indexes such as collagen deposition and airway smooth muscle cell proliferation, while ESI-09 increased airway inflammation and airway remodeling. In vitro cultured mice ASMCs, 8pCPT dose-dependently inhibited, whereas ESI-09 promoted ASMCs proliferation. Interestingly, 8pCPT promoted the apoptosis of ASMCs, whereas ESI-09 had no effect on ASMCs apoptosis. Lastly, confocal Ca2+ fluorescence examination found that 8pCPT could inhibit SOCE in ASMCs at 100 µM, and ESI-09 promoted SOCE of ASMCs at 10 µM and 100 µM. In addition, the promoting effect of ESI-09 on ASMCs proliferation was inhibited by store-operated Ca2+ channel blocker, SKF-96365. CONCLUSIONS: Our results suggest that Epac has a protecting effect on asthmatic airway inflammation and airway remodeling, and Epac reduces ASMCs proliferation by inhibiting SOCE in part.

The Epac1 Protein: Pharmacological Modulators, Cardiac Signalosome and Pathophysiology.

The second messenger 3',5'-cyclic adenosine monophosphate (cAMP) is one of the most important signalling molecules in the heart as it regulates many physiological and pathophysiological processes. In addition to the classical protein kinase A (PKA) signalling route, the exchange proteins directly activated by cAMP (Epac) mediate the intracellular functions of cAMP and are now emerging as a new key cAMP effector in cardiac pathophysiology. In this review, we provide a perspective on recent advances in the discovery of new chemical entities targeting the Epac1 isoform and illustrate their use to study the Epac1 signalosome and functional characterisation in cardiac cells. We summarize the role of Epac1 in different subcompartments of the cardiomyocyte and discuss how cAMP-Epac1 specific signalling networks may contribute to the development of cardiac diseases. We also highlight ongoing work on the therapeutic potential of Epac1-selective small molecules for the treatment of cardiac disorders.

Overexpression of EPAC2 reduces the invasion of glioma cells via MMP-2.

Exchange proteins directly activated by cAMP (EPACs) are crucial cyclic adenosine 3',5'-monophosphate- determined signaling pathway intercessors, which are associated with the pathogenesis of neurological disorders and numerous human diseases. To the best of our knowledge, the role of EPAC2 signaling via matrix metalloproteinase 2 (MMP-2) in the pathogenesis of glioma has not been studied. Therefore, the present study focused on the role of EPAC2 in glioma, and assessed the invasiveness of human glioma cell lines following EPAC2 overexpression. Expression levels of EPAC2 in normal brain tissues and clinical glioma specimens were detected by western blotting. An EPAC2 overexpression vector was transfected into U251 and U87 cell lines to increase the expression levels of EPAC2. Expression levels of MMP-2 were detected by western blotting, and the invasive abilities of glioma cells were detected by a Transwell assay. EPAC2 was relatively highly expressed in normal brain tissue, while EPAC2 expression was significantly decreased in clinical glioma specimens (P<0.01). In vitro transfection of EPAC2 overexpression vector significantly reduced the MMP-2 protein levels of glioma cells, and, at the same time, the invasive cell number was significantly decreased in a Transwell assay. The present study demonstrated that MMP-2 regulation via EPAC2 overexpression is a novel promising therapeutic route in malignant types of glioma.

Exchange Protein Directly Activated by cAMP Modulates Ebola Virus Uptake into Vascular Endothelial Cells.

Members of the family Filoviridae, including Ebola virus (EBOV) and Marburg virus (MARV), cause severe hemorrhagic fever in humans and nonhuman primates. Given their high lethality, a comprehensive understanding of filoviral pathogenesis is urgently needed. In the present studies, we revealed that the exchange protein directly activated by cAMP 1 (EPAC1) gene deletion protects vasculature in ex vivo explants from EBOV infection. Importantly, pharmacological inhibition of EPAC1 using EPAC-specific inhibitors (ESIs) mimicked the EPAC1 knockout phenotype in the ex vivo model. ESI treatment dramatically decreased EBOV infectivity in both ex vivo vasculature and in vitro vascular endothelial cells (ECs). Furthermore, postexposure protection of ECs against EBOV infection was conferred using ESIs. Protective efficacy of ESIs in ECs was observed also in MARV infection. Additional studies using a vesicular stomatitis virus pseudotype that expresses EBOV glycoprotein (EGP-VSV) confirmed that ESIs reduced infection in ECs. Ultrastructural studies suggested that ESIs blocked EGP-VSV internalization via inhibition of macropinocytosis. The inactivation of EPAC1 affects the early stage of viral entry after viral binding to the cell surface, but before early endosome formation, in a phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)-dependent manner. Our study delineated a new critical role of EPAC1 during EBOV uptake into ECs.

Alleviation of thalamic pain by cilostazol administration: a case report.

Thalamic pain is severe and treatment-resistant; however, there are few available options for improving thalamic pain. This study demonstrated that thalamic pain was alleviated by administration of cilostazol, suggesting that cilostazol may be a candidate for treating thalamic pain.

Compartmentalized crosstalk of CFTR and TMEM16A (ANO1) through EPAC1 and ADCY1.

Airway epithelial cells express both Ca2+ activated TMEM16A/ANO1 and cAMP activated CFTR anion channels. Previous work suggested a significant crosstalk of intracellular Ca2+ and cAMP signaling pathways, leading to activation of both chloride channels. We demonstrate that in airway epithelial cells, stimulation of purinergic or muscarinic G-protein coupled receptors (GPCRs) activates TMEM16A and CFTR. Additional expression of Gq/11 and phospholipase C coupled GPCRs strongly enhanced the crosstalk between Ca2+- and cAMP-dependent signaling. Knockdown of endogenous GRCRs attenuated crosstalk and functional coupling between TMEM16A and CFTR. The number of receptors did not affect expression or membrane localization of TMEM16A or CFTR, but controlled assembly of the local signalosome. GPCRs translocate Ca2+-sensitive adenylate cyclase type 1 (ADCY1) and exchange protein directly activated by cAMP (EPAC1) to particular plasma membrane domains containing GPCRs, CFTR and TMEM16A, thereby producing compartmentalized Ca2+ and cAMP signals and significant crosstalk. While biosynthesis and membrane trafficking of CFTR requires a functional Golgi apparatus, maturation and membrane trafficking of TMEM16A may occur independent of the Golgi. Because Ca2+ activated TMEM16A currents are only transient, continuous Cl- secretion by airway epithelial cells requires CFTR. The present data also explain why receptor-dependent activation of TMEM16A is more efficient than direct stimulation by Ca2+.

Depotentiation of intact rat cardiac muscle unmasks an Epac-dependent increase in myofilament Ca(2+) sensitivity.

Recently, a family of guanine nucleotide exchange factors have been identified in many cell types as important effectors of cyclic adenosine 3',5'-monophospahte (cAMP) signalling that is independent of protein kinase A (PKA). In the heart, investigation of exchange protein directly activated by cAMP (Epac) has yielded conflicting results. Since cAMP is an important regulator of cardiac contractility, this study aimed to examine whether Epac activation modulates excitation-contraction coupling in ventricular preparations from rat hearts. The study used 8-(4-chlorophenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate (cpTOME), an analogue of cAMP that activates Epac, but not PKA. In isolated myocytes, cpTOME increased Ca(2+) spark frequency from about 7 to 32/100 µm(3)/s (n = 10), P = 0.05 with a reduction in the peak amplitude of the sparks. Simultaneous measurements of intracellular Ca(2+) and isometric force in multicellular trabeculae (n = 7, 1.5 mmol/L [Ca(2+)]o) revealed no effect of Epac activation on either the amplitude of Ca(2+) transients (Control 0.7 ± 0.1 vs cpTOME 0.7 ± 0.1; 340/380 fura-2 ratio, P = 0.35) or on peak stress (Control 24 ± 5 mN/mm(2) vs cpTOME 23 ± 5 mN/mm(2), P = 0.20). However, an effect of Epac in trabeculae was unmasked by lowering extracellular [Ca(2+)]o. In these depotentiated trabeculae, activation of the Epac pathway increased myofilament Ca(2+) sensitivity, an effect that was blocked by addition of KN-93, a Ca(2+)/calmodulin-dependent protein kinase II (CaMK-II) inhibitor. This study suggests that Epac activation may be a useful therapeutic target to increase the strength of contraction during low inotropic states.

Ca2+ influx through L-type Ca2+ channels and Ca2+-induced Ca2+ release regulate cAMP accumulation and Epac1-dependent ERK 1/2 activation in INS-1 cells.

We previously reported that INS-1 cells expressing the intracellular II-III loop of the L-type Ca(2+) channel Cav1.2 (Cav1.2/II-III cells) are deficient in Ca(2+)-induced Ca(2+) release (CICR). Here we show that glucose-stimulated ERK 1/2 phosphorylation (GSEP) is slowed and reduced in Cav1.2/II-III cells compared to INS-1 cells. This parallels a decrease in glucose-stimulated cAMP accumulation (GS-cAMP) in Cav1.2/II-III cells. Influx of Ca(2+) via L-type Ca(2+) channels and CICR play roles in both GSEP and GS-cAMP in INS-1 cells since both are inhibited by nicardipine or ryanodine. Further, the Epac1-selective inhibitor CE3F4 abolishes glucose-stimulated ERK activation in INS-1 cells, as measured using the FRET-based sensor EKAR. The non-selective Epac antagonist ESI-09 but not the Epac2-selective antagonist ESI-05 nor the PKA antagonist Rp-cAMPs inhibits GSEP in both INS-1 and Cav1.2/II-III cells. We conclude that L-type Ca(2+) channel-dependent cAMP accumulation, that's amplified by CICR, activates Epac1 and drives GSEP in INS-1 cells.

EPAC2-mediated calreticulin regulates LIF and COX2 expression in human endometrial glandular cells.

The proper production of the implantation-related factors, leukemia inhibitory factor (LIF), cyclooxygenase 2 (COX2, PTGS2), and prostaglandin E2 (PGE2) in the uterine glands is essential for embryo implantation and the establishment of endometrial receptivity. It has been shown that cAMP-mediated protein kinase A (PKA) signaling regulates the production of these factors. We have previously reported that exchange protein directly activated by cAMP 2 (EPAC2, RAPGEF4), another cAMP mediator, is involved in the differentiation of endometrial stromal cells through the regulation of the expression of calreticulin (CALR). To address whether EPAC2-CALR signaling is involved in the expression of implantation-related factors, we examined the effect of EPAC2 and CALR knockdown on their expression in cultured human endometrial glandular epithelial EM1 cells, treated with forskolin, an adenylyl cyclase activator, an EPAC-selective cAMP analog (8-(4-chlorophenylthio)-2'-O-methyl cAMP (CPT)), or a PKA-selective cAMP analog (N(6)-phenyl-cAMP (Phe)). In addition, the status of cell senescence was examined. EPAC2 knockdown suppressed the expression of CALR protein and mRNA in EM1 cells. Forskolin- or Phe-, but not CPT-, induced expression of LIF or PTGS2 and secretion of PGE2 was inhibited in EPAC2- or CALR-silenced EM1 cells. In addition, knockdown of EPAC2 or CALR increased senescence-associated beta galactosidase activity and expression of p21 but decreased expression of p53. These findings indicate that expression of CALR regulated by EPAC2 in endometrial glandular epithelial cells is critical for the expression of LIF and PTGS2-mediated production of PGE2 through cAMP signaling. Furthermore, EPAC2 and CALR could play a role in the maintenance of gland function.

Cyclic AMP sensor EPAC proteins and energy homeostasis.

The pleiotropic second-messenger cAMP plays a crucial role in mediating the effects of various hormones on metabolism. The major intracellular functions of cAMP are transduced by protein kinase A (PKA) and by exchange proteins directly activated by cAMP (EPACs). The latter act as guanine-nucleotide exchange factors for the RAS-like small G proteins Rap1 and Rap2. Although the role of PKA in regulating energy balance has been extensively studied, the impact of EPACs remains relatively enigmatic. This review summarizes recent genetic and pharmacological studies concerning EPAC involvement in glucose homeostasis and energy balance via the regulation of leptin and insulin signaling pathways. In addition, the development of small-molecule EPAC-specific modulators and their therapeutic potential for the treatment of diabetes and obesity are discussed.

Calcium/calmodulin-dependent serine protein kinase is involved in exendin-4-induced insulin secretion in INS-1 cells.

OBJECTIVE: Exendin-4 (Ex-4) is an anti-diabetic drug that is a potent agonist of the glucagon-like peptide-1 (GLP-1) receptor. It has already been approved for the treatment of type 2 diabetes mellitus, but its underlying mechanisms of action are not fully understood. Calcium/calmodulin-dependent serine protein kinase (CASK), which plays a vital role in the transport and release of neurotransmitters in neurons, is expressed in pancreatic islet cells and ß-cells. This study aimed to investigate whether CASK is involved in the insulin secretagogue action induced by Ex-4 in INS-1 cells. MATERIAL/METHODS: A glucose-stimulated insulin secretion (GSIS) assay was performed with or without siRNA treatment against CASK. The expression level and location of CASK were evaluated by real-time PCR, western blotting and immunofluorescence. With the use of a protein kinase A (PKA) inhibitor or an exchange protein directly activated by cAMP-2 (Epac2) agonist, immunoblotting was performed to establish the signaling pathway through which Ex-4 alters CASK expression. RESULTS: Knock-down of CASK significantly attenuated the Ex-4-enhanced insulin release, and we showed that Ex-4 could increase transcription of CASK mRNA and expression of CASK protein but did not change the cellular location of CASK. A PKA inhibitor reduced the ability of Ex-4 to stimulate CASK expression, but an Epac2 agonist had no effect suggesting that regulation was mediated by the cAMP/PKA pathway. CONCLUSION: Our study suggests that the stimulation of ß-cell insulin secretion by Ex-4 is mediated, at least in part, by CASK via a novel signaling mechanism.