Presynaptic effectors contributing to cAMP-induced synaptic potentiation in Drosophila

cAMP analogs and activation of adenylyl cyclase by forskolin strongly potentiate synaptic transmission at the Drosophila neuromuscular junction. These effects are generally attributed to activation of cAMP-dependent protein kinase. Recent reports on crustacean and mammalian synapses have implicated other cAMP-dependent effectors in synaptic potentiation. Drosophila neuromuscular junctions were tested for effects of two known cAMP-dependent effectors: hyperpolarization-activated, cyclic nucleotide-regulated channels (HCNCs) and guanine nucleotide exchange protein activated by cAMP (Epac). Forskolin-induced enhancement of synaptic transmission was drastically reduced by a blocker of HCNCs, but not completely eliminated. A specific agonist for Epac modestly enhanced synaptic potentials. This agonist also stabilized their amplitudes in the presence of a blocker of HCNCs. The observations implicate HCNCs and Epac in cAMP-dependent potentiation that does not require cAMP-dependent protein kinase, indicating that additional previously unexplored factors contribute to synaptic plasticity in Drosophila. Genetic and molecular techniques available for Drosophila can be used to define the underlying molecular basis for cAMP-dependent synaptic potentiation.     

β-Adrenergic Receptors Activate Exchange Protein Directly Activated by cAMP (Epac), Translocate Munc13-1, and Enhance the Rab3A-RIM1α Interaction to Potentiate Glutamate Release at Cerebrocortical Nerve Terminals

The adenylyl cyclase activator forskolin facilitates synaptic transmission presynaptically via cAMP-dependent protein kinase (PKA). In addition, cAMP also increases glutamate release via PKA-independent mechanisms, although the downstream presynaptic targets remain largely unknown. Here, we describe the isolation of a PKA-independent component of glutamate release in cerebrocortical nerve terminals after blocking Na⁺ channels with tetrodotoxin. We found that 8-pCPT-2′-O-Me-cAMP, a specific activator of the exchange protein directly activated by cAMP (Epac), mimicked and occluded forskolin-induced potentiation of glutamate release. This Epac-mediated increase in glutamate release was dependent on phospholipase C, and it increased the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Moreover, the potentiation of glutamate release by Epac was independent of protein kinase C, although it was attenuated by the diacylglycerol-binding site antagonist calphostin C. Epac activation translocated the active zone protein Munc13-1 from soluble to particulate fractions; it increased the association between Rab3A and RIM1α and redistributed synaptic vesicles closer to the presynaptic membrane. Furthermore, these responses were mimicked by the β-adrenergic receptor (βAR) agonist isoproterenol, consistent with the immunoelectron microscopy and immunocytochemical data demonstrating presynaptic expression of βARs in a subset of glutamatergic synapses in the cerebral cortex. Based on these findings, we conclude that βARs couple to a cAMP/Epac/PLC/Munc13/Rab3/RIM-dependent pathway to enhance glutamate release at cerebrocortical nerve terminals.     

Rap-linked cAMP signaling Epac proteins: compartmentation, functioning and disease implications

Epac proteins respond to the second messenger cyclic AMP (cAMP) and are activated by Gs coupled receptors. They act as specific guanine nucleotide exchange factors (GEFs) for the small G proteins, Rap1 and Rap2 of the Ras family. A plethora of studies using 8-pCPT-2′-O-Me-cAMP, an Epac agonist, has revealed the importance of these multi-domain proteins in the control of key cellular functions such as cell division, migration, growth and secretion. Epac and protein kinase A (PKA) may act independently but are often associated with the same biological process, in which they fulfill either synergistic or opposite effects. In addition, compelling evidence is now accumulating about the formation of molecular complexes in distinct cellular compartments that influence Epac signaling and cellular function. Epac is spatially and temporally regulated by scaffold protein and its effectors are interconnected with other signaling pathways. Pathophysiological changes in Epac signaling may underlie certain diseases.     

Cooperation of Epac1/Rap1/Akt and PKA in prostaglandin E(2) -induced proliferation of human umbilical cord blood derived mesenchymal stem cells: Involvement of c-Myc and VEGF expression

Prostaglandin E₂ (PGE₂) is well known to regulate cell functions through cAMP; however, the role of exchange protein directly activated by cAMP (Epac1) and protein kinase A (PKA) in modulating such functions is unknown in human umbilical cord blood‐derived mesenchymal stem cells (hUCB‐MSCs). Therefore, we investigated the relationship between Epac1 and PKA during PGE₂‐induced hUCB‐MSC proliferation and its related signaling pathways. PGE₂ increased cell proliferation, and E‐type prostaglandin (EP) 2 receptor mRNA expression level and activated cAMP generation, which were blocked by EP2 receptor selective antagonist AH 6809. PGE₂ increased Epac1 expression, Ras‐related protein 1 (Rap1) activation level, and Akt phosphorylation, which were inhibited by AH 6809, adenylyl cyclase inhibitor SQ 22536, and Epac1/Rap1‐specific siRNA. Also, PGE₂ increased PKA activity, which was inhibited by AH 6809, SQ 22536, and PKA inhibitor PKI. HUCB‐MSCs were incubated with the Epac agonist 8‐pCPT‐cAMP or the PKA agonist 6‐phe‐cAMP to examine whether Epac1/Rap1/Akt activation was independent of PKA activation. 8‐pCPT‐cAMP increased Akt phosphorylation but not PKA activity. 6‐Phe‐cAMP increased PKA activity, but not Akt phosphorylation. Additionally, an Akt inhibitor or PKA inhibitor (PKI) did not block the PGE₂‐induced increase in PKA activity or Akt phosphorylation, respectively. Moreover, PGE₂ increased glycogen synthase kinase (GSK)‐3β phosphorylation and nuclear translocation of active‐β‐catenin, which were inhibited by Akt inhibitor or/and PKI. PGE₂ increased c‐Myc and vascular endothelial growth factor (VEGF) expression levels, which were blocked by β‐catenin siRNA. In conclusion, PGE₂ stimulated hUCB‐MSC proliferation through β‐catenin‐mediated c‐Myc and VEGF expression via Epac/Rap1/Akt and PKA cooperation. J. Cell. Physiol. 227: 3756–3767, 2012. © 2012 Wiley Periodicals, Inc.     

Cyclic AMP induces integrin-mediated cell adhesion through Epac and Rap1 upon stimulation of the beta 2-adrenergic receptor

cAMP controls many cellular processes mainly through the activation of protein kinase A (PKA). However, more recently PKA-independent pathways have been established through the exchange protein directly activated by cAMP (Epac), a guanine nucleotide exchange factor for the small GTPases Rap1 and Rap2. In this report, we show that cAMP can induce integrin-mediated cell adhesion through Epac and Rap1. Indeed, when Ovcar3 cells were treated with cAMP, cells adhered more rapidly to fibronectin. This cAMP effect was insensitive to the PKA inhibitor H-89. A similar increase was observed when the cells were transfected with Epac. Both the cAMP effect and the Epac effect on cell adhesion were abolished by the expression of Rap1-GTPase-activating protein, indicating the involvement of Rap1 in the signaling pathway. Importantly, a recently characterized cAMP analogue, 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate, which specifically activates Epac but not PKA, induced Rap-dependent cell adhesion. Finally, we demonstrate that external stimuli of cAMP signaling, i.e., isoproterenol, which activates the G alpha s-coupled beta 2-adrenergic receptor can induce integrin-mediated cell adhesion through the Epac-Rap1 pathway. From these results we conclude that cAMP mediates receptor-induced integrin-mediated cell adhesion to fibronectin through the Epac-Rap1 signaling pathway.     

The (R)-enantiomer of CE3F4 is a preferential inhibitor of human exchange protein directly activated by cyclic AMP isoform 1 (Epac1)

Isoform 1 and isoform 2 of exchange protein directly activated by cAMP (Epac1 and Epac2) contribute to cAMP signaling in numerous cellular processes. Their guanine-nucleotide exchange factor (GEF) activity toward the small GTP-binding protein Rap1 is stimulated by the agonist cAMP. CE3F4, a tetrahydroquinoline analog, prevents Epac1 activation in vitro and in living cultured cells by inhibiting the GEF activity of Epac1. However, the activity of the (R)- and (S)-enantiomers of CE3F4, as well as the ability of CE3F4 and its analogs to inhibit Epac2 GEF activity, have not yet been investigated. In this study, we report that (R)-CE3F4 is a more potent cAMP antagonist than racemic CE3F4 and (S)-CE3F4, inhibiting the GEF activity of Epac1 with 10-times more efficiency than (S)-CE3F4. Epac2, in contrast to Epac1, is activated more efficiently by cAMP than by 8-(4-chlorophenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (007), an Epac-selective cAMP analog. (R)-CE3F4 displays Epac isoform preference, with 10-fold selectivity for Epac1 over Epac2. Deletion of the N-terminal cyclic nucleotide-binding domain of Epac2 does not affect the characteristics of activation of Epac2 by cAMP and by 007, nor its inhibition by CE3F4. Finally, the evaluation of a series of CE3F4 structural analogs as GEF inhibitors allowed identifying structural features that are important for high Epac1 inhibitory activity of CE3F4. We conclude that the (R)-enantiomer of CE3F4 is a preferential inhibitor of Epac1 with high potency in the low micromolar range, and we suggest that this compound may be a useful pharmacological tool for investigating the functional role of Epac1 in cAMP signaling.     

The cAMP-activated GTP exchange factor, Epac1 upregulates plasma membrane and nuclear Akt kinase activities in 8-CPT-2-O-Me-cAMP-stimulated macrophages: Gene silencing of the cAMP-activated GTP exchange Epac1 prevents 8-CPT-2-O-Me-cAMP activation of Akt activity in macrophages

cAMP regulates a wide range of processes through its downstream effectors including PKA, and the family of guanine nucleotide exchange factors. Depending on the cell type, cAMP inhibits or stimulates growth and proliferation in a PKA-dependent or independent manner. PKA-independent effects are mediated by PI 3-kinases-Akt signaling and EPAC1 (exchange protein directly activated by cAMP) activation. Recently, we reported PKA-independent activation of the protein kinase Akt as well co-immunoprecipitation of Epac1 with Rap1, p-Akt(Thr-308), and p-Akt(Ser-473) in forskolin-stimulated macrophages. To further probe the role of Epac1 in Akt protein kinase activation and cellular proliferation, we employed the cAMP analog 8-CPT-2-O-Me-cAMP, which selectively binds to Epac1 and triggers Epac1 signaling. We show the association of Epac1 with activated Akt kinases by co-immunoprecipitation and GST-pulldown assays. Silencing Epac1 gene expression by RNA interference significantly reduced levels of Epac1 mRNA, Epac protein, Rap1 GTP, p-ERK1/2, p-B-Raf, p110alpha catalytic subunit of PI 3-kinase, p-PDK, and p-p(70s6k). Silencing Epac1 gene expression by RNA interference also suppressed 8-CPT-2-O-Me-cAMP-upregulated protein and DNA synthesis. Concomitantly, 8-CPT-2-O-Me-cAMP-mediated upregulation of Akt(Thr-308) protein kinase activity and p-Akt(Thr-308) levels was prevented in plasma membranes and nuclei of the cells. In contrast, silencing Epac1 gene expression reduced Akt(Ser-473) kinase activity and p-Akt(Ser-473) levels in plasma membranes, but showed negligible effects on nuclear activity. In conclusion, we show that cAMP-induced Akt kinase activation and cellular proliferation is mediated by Epac1 which appears to function as an accessory protein for Akt activation.     

β‐Guanidinopropionic acid extends the lifespan of Drosophila melanogaster via an AMP‐activated protein kinase‐dependent increase in autophagy

Previous studies have demonstrated that AMP‐activated protein kinase (AMPK) controls autophagy through the mammalian target of rapamycin (mTOR) and Unc‐51 like kinase 1 (ULK1/Atg1) signaling, which augments the quality of cellular housekeeping, and that β‐guanidinopropionic acid (β‐GPA), a creatine analog, leads to a chronic activation of AMPK. However, the relationship between β‐GPA and aging remains elusive. In this study, we hypothesized that feeding β‐GPA to adult Drosophila produces the lifespan extension via activation of AMPK‐dependent autophagy. It was found that dietary administration of β‐GPA at a concentration higher than 900 mm induced a significant extension of the lifespan of Drosophila melanogaster in repeated experiments. Furthermore, we found that Atg8 protein, the homolog of microtubule‐associated protein 1A/1B‐light chain 3 (LC3) and a biomarker of autophagy in Drosophila, was significantly upregulated by β‐GPA treatment, indicating that autophagic activity plays a role in the effect of β‐GPA. On the other hand, when the expression of Atg5 protein, an essential protein for autophagy, was reduced by RNA interference (RNAi), the effect of β‐GPA on lifespan extension was abolished. Moreover, we found that AMPK was also involved in this process. β‐GPA treatment significantly elevated the expression of phospho‐T172‐AMPK levels, while inhibition of AMPK by either AMPK‐RNAi or compound C significantly attenuated the expression of autophagy‐related proteins and lifespan extension in Drosophila. Taken together, our results suggest that β‐GPA can induce an extension of the lifespan of Drosophila via AMPK‐Atg1‐autophagy signaling pathway.     

Epac and PKA: a tale of two intracellular cAMP receptors

cAMP-mediated signaling pathways regulate a multitude of important biological processes under both physiological and pathological conditions, including diabetes, heart failure and cancer. In eukaryotic cells, the effects of cAMP are mediated by two ubiquitously expressed intracellular cAMP receptors, the classic protein kinase A (PKA)/cAMP-dependent protein kinase and the recently discovered exchange protein directly activated by cAMP (Epac)/cAMP-regulated guanine nucleotide exchange factors. Like PKA, Epac contains an evolutionally conserved cAMP binding domain that acts as a molecular switch for sensing intracellular second messenger cAMP levels to control diverse biological functions. The existence of two families of cAMP effectors provides a mechanism for a more precise and integrated control of the cAMP signaling pathways in a spatial and temporal manner. Depending upon the specific cellular environments as well as their relative abundance, distribution and localization, Epac and PKA may act independently, converge synergistically or oppose each other in regulating a specific cellular function.     

Jelly belly trans‐synaptic signaling to anaplastic lymphoma kinase regulates neurotransmission strength and synapse architecture

In Drosophila, the secreted signaling molecule Jelly Belly (Jeb) activates anaplastic lymphoma kinase (Alk), a receptor tyrosine kinase, in multiple developmental and adult contexts. We have shown previously that Jeb and Alk are highly enriched at Drosophila synapses within the CNS neuropil and neuromuscular junction (NMJ) and postulated a conserved intercellular signaling function. At the embryonic and larval NMJ, Jeb is localized in the motor neuron presynaptic terminal whereas Alk is concentrated in the muscle postsynaptic domain surrounding boutons, consistent with anterograde trans‐synaptic signaling. Here, we show that neurotransmission is regulated by Jeb secretion by functional inhibition of Jeb–Alk signaling. Jeb is a novel negative regulator of neuromuscular transmission. Reduction or inhibition of Alk function results in enhanced synaptic transmission. Activation of Alk conversely inhibits synaptic transmission. Restoration of wild‐type postsynaptic Alk expression in Alk partial loss‐of‐function mutants rescues NMJ transmission phenotypes and confirms that postsynaptic Alk regulates NMJ transmission. The effects of impaired Alk signaling on neurotransmission are observed in the absence of associated changes in NMJ structure. Complete removal of Jeb in motor neurons, however, disrupts both presynaptic bouton architecture and postsynaptic differentiation. Nonphysiologic activation of Alk signaling also negatively regulates NMJ growth. Activation of Jeb–Alk signaling triggers the Ras‐MAP kinase cascade in both pre‐ and postsynaptic compartments. These novel roles for Jeb–Alk signaling in the modulation of synaptic function and structure have potential implications for recently reported Alk functions in human addiction, retention of spatial memory, cognitive dysfunction in neurofibromatosis, and pathogenesis of amyotrophic lateral sclerosis. © 2012 Wiley Periodicals, Inc. Develop Neurobiol, 2013     

Epac activation converts cAMP from a proliferative into a differentiation signal in PC12 cells

Elevation of the intracellular cAMP concentration ([cAMP]i) regulates metabolism, cell proliferation, and differentiation and plays roles in memory formation and neoplastic growth. cAMP mediates its effects mainly through activation of protein kinase A (PKA) as well as Epac1 and Epac2, exchange factors activating the small GTPases Rap1 and Rap2. However, how cAMP utilizes these effectors to induce distinct biological responses is unknown. We here studied the specific roles of PKA and Epac in neuroendocrine PC12 cells. In these cells, elevation of [cAMP]i activates extracellular signal-regulated kinase (ERK) 1/2 and induces low-degree neurite outgrowth. The present study showed that specific stimulation of PKA triggered ERK1/2 activation that was considerably more transient than that observed upon simultaneous activation of both PKA and Epac. Unexpectedly, the PKA-specific cAMP analog induced cell proliferation rather than neurite outgrowth. The proliferative signaling pathway activated by the PKA-specific cAMP analog involved activation of the epidermal growth factor receptor and ERK1/2. Activation of Epac appeared to extend the duration of PKA-dependent ERK1/2 activation and converted cAMP from a proliferative into an anti-proliferative, neurite outgrowth-promoting signal. Thus, the present study showed that the outcome of cAMP signaling can depend heavily on the set of cAMP effectors activated.     

β1‐adrenergic receptor antagonists signal via PDE4 translocation

It is generally assumed that antagonists of G_s‐coupled receptors do not activate cAMP signalling, because they do not stimulate cAMP production via G_s‐protein/adenylyl cyclase activation. Here, we report a new signalling pathway whereby antagonists of β₁‐adrenergic receptors (β₁ARs) increase cAMP levels locally without stimulating cAMP production directly. Binding of antagonists causes dissociation of a preformed complex between β₁ARs and Type‐4 cyclic nucleotide phosphodiesterases (PDE4s). This reduces the local concentration of cAMP‐hydrolytic activity, thereby increasing submembrane cAMP and PKA activity. Our study identifies receptor/PDE4 complex dissociation as a novel mechanism of antagonist action that contributes to the pharmacological properties of β₁AR antagonists and might be shared by other receptor subtypes.     

Off-target effect of the Epac agonist 8-pCPT-2′-O-Me-cAMP on P2Y12 receptors in blood platelets

The primary target of the cAMP analogue 8-pCPT-2′-O-Me-cAMP is exchange protein directly activated by cAMP (Epac). Here we tested potential off-target effects of the Epac activator on blood platelet activation signalling. We found that the Epac analogue 8-pCPT-2′-O-Me-cAMP inhibits agonist-induced-GPCR-stimulated, but not collagen-stimulated, P-selectin surface expression on Epac1 deficient platelets. In human platelets, 8-pCPT-2′-O-Me-cAMP inhibited P-selectin expression elicited by the PKC activator PMA. This effect was abolished in the presence of the extracellular ADP scavenger system CP/CPK. In silico modelling of 8-pCPT-2′O-Me-cAMP binding into the purinergic platelet receptor P2Y₁₂ revealed that the analogue docks similar to the P2Y₁₂ antagonist 2MeSAMP. The 8-pCPT-2′-O-Me-cAMP analogue per se, did not provoke Rap 1 (Rap 1-GTP) activation or phosphorylation on the vasodilator-stimulated phosphoprotein (VASP) at Ser-157. In addition, the protein kinase A (PKA) antagonists Rp-cAMPS and Rp-8-Br-cAMPS failed to block the inhibitory effect of 8-pCPT-2′-O-Me-cAMP on thrombin- and TRAP-induced Rap 1 activation, thus suggesting that PKA is not involved. We conclude that the 8-pCPT-2′-O-Me-cAMP analogue is able to inhibit agonist-induced-GPCR-stimulated P-selectin independent from Epac1; the off-target effect of the analogue appears to be mediated by antagonistic P2Y₁₂ receptor binding. This has implications when using cAMP analogues on specialised system involving such receptors. We found, however that the Epac agonist 8-Br-2′-O-Me-cAMP did not affect platelet activation at similar concentrations.     

The thrombin receptor mediates functional activity‐dependent neuromuscular synapse reduction via protein kinase C activation in vitro

Activity‐dependent selective reduction of synaptic efficacy is expressed in an in vitro system involving mouse spinal cord and muscle cells. Thrombin or electrical stimulation of the innervating axons induces a decrease in neuromuscular synapse strength, and a specific thrombin inhibitor, hirudin, blocks the electrically evoked down‐regulation of synapse effectiveness. We further demonstrate that a thrombin receptor‐activating peptide (TRAP), SFLLRNPNDKYEPF, produces a decrement of synapse strength. Both TRAP and electrically evoked synapse decrement are prevented by the specific protein kinase C blocker calphostin C, and the TRAP‐evoked synapse decrement is unaffected by a specific protein kinase A blocker, H‐89. Thus, we propose that muscle activity, thrombin release, and thrombin receptor and PKC activation are initial steps in the process of the activity‐dependent synapse reduction expressed in our system. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 369–381, 1999     

Rap1-mediated activation of extracellular signal-regulated kinases by cyclic AMP is dependent on the mode of Rap1 activation

Like other small G proteins of the Ras superfamily, Rap1 is activated by distinct guanine nucleotide exchange factors (GEFs) in response to different signals to elicit cellular responses. Activation of Rap1 by cyclic AMP (cAMP) can occur via cAMP-dependent protein kinase A (PKA)-independent and PKA-dependent mechanisms. PKA-independent activation of Rap1 by cAMP is mediated by direct binding of cAMP to Rap1-guanine nucleotide exchange factors (Rap1-GEFs) Epac1 (exchange protein directly activated by cAMP 1) and Epac2 (Epac1 and Epac2 are also called cAMP-GEFI and -GEFII). The availability of cAMP analogues that selectively activate Epacs, but not PKA, provides a specific tool to activate Rap1. It has been argued that the inability of these analogues to regulate extracellular signal-regulated kinases (ERKs) signaling despite activating Rap1 provides evidence that Rap1 is incapable of regulating ERKs. We confirm that the PKA-independent activation of Rap1 by Epac1 activates a perinuclear pool of Rap1 and that this does not result in ERK activation. However, we demonstrate that this inability to regulate ERKs is not a property of Rap1 but is rather a property of Epacs themselves. The addition of a membrane-targeting motif to Epac1 (Epac-CAAX) relocalizes Epac1 from its normal perinuclear locale to the plasma membrane. In this new locale it is capable of activating ERKs in a Rap1- and cAMP-dependent manner. Rap1 activation by Epac-CAAX, but not wild-type Epac, triggers its association with B-Raf. Therefore, we propose that its intracellular localization prevents Epac1 from activating ERKs. C3G (Crk SH3 domain Guanine nucleotide exchanger) is a Rap1 exchanger that is targeted to the plasma membrane upon activation. We show that C3G can be localized to the plasma membrane by cAMP/PKA, as can Rap1 when activated by cAMP/PKA. Using a small interfering RNA approach, we demonstrate that C3G is required for the activation of ERKs and Rap1 by cAMP/PKA. This activation requires the GTP-dependent association of Rap1 with B-Raf. These data demonstrate that B-Raf is a physiological target of Rap1, but its utilization as a Rap1 effector is GEF specific. We propose a model that specific GEFs activate distinct pools of Rap1 that are differentially coupled to downstream effectors.     

Parathyroid hormone 2 receptor is a functional marker of nociceptive myelinated fibers responsible for neuropathic pain

We have previously demonstrated that parathyroid hormone 2 (PTH2) receptors are expressed in dorsal root ganglion (DRG) neurons and that its endogenous agonist tuberoinfundibular peptide of 39 residues (TIP39) causes nociceptive paw flexor responses after intraplantar administration. Here we found that the PTH2 receptor is selectively localized on myelinated A‐, but not unmyelinated C‐fibers using immunohistochemical labeling, based on PTH2 receptor expression on antibody N52‐positive medium/large‐sized DRG neurons, but not on TRPV1, substance P, P2X₃ receptor or isolectin B4‐binding protein‐positive small‐sized DRG neurons. Pharmacological studies showed that TIP39‐induced nociceptive responses were mediated by activation of G_s and cAMP‐dependent protein kinase. We also found that nociceptive responses induced by TIP39‐ or the cAMP analog 8‐bromo‐cAMP were significantly greater following partial sciatic nerve injury induced neuropathic pain, without changes in PTH2 receptor expression. Together these data suggest that activation of PTH2 receptors stimulates nociceptive A‐fiber through G_s‐cAMP‐dependent protein kinase signaling, and this pathway has elevated sensitization following nerve injury.     

Epac as a novel effector of airway smooth muscle relaxation

Dysfunctional regulation of airway smooth muscle tone is a feature of obstructive airway diseases such as asthma and chronic obstructive pulmonary disease. Airway smooth muscle contraction is directly associated with changes in the phosphorylation of myosin light chain (MLC), which is increased by Rho and decreased by Rac. Although cyclic adenosine monophosphate (cAMP)‐elevating agents are believed to relieve bronchoconstriction mainly via activation of protein kinase A (PKA), here we addressed the role of the novel cAMP‐mediated exchange protein Epac in the regulation of airway smooth muscle tone. Isometric tension measurements showed that specific activation of Epac led to relaxation of guinea pig tracheal preparations pre‐contracted with methacholine, independently of PKA. In airway smooth muscle cells, Epac activation reduced methacholine‐induced MLC phosphorylation. Moreover, when Epac was stimulated, we observed a decreased methacholine‐induced RhoA activation, measured by both stress fibre formation and pull‐down assay whereas the same Epac activation prevented methacholine‐induced Rac1 inhibition measured by pull‐down assay. Epac‐driven inhibition of both methacholine‐induced muscle contraction by Toxin B‐1470, and MLC phosphorylation by the Rac1‐inhibitor NSC23766, were significantly attenuated, confirming the importance of Rac1 in Epac‐mediated relaxation. Importantly, human airway smooth muscle tissue also expresses Epac, and Epac activation both relaxed pre‐contracted human tracheal preparations and decreased MLC phosphorylation. Collectively, we show that activation of Epac relaxes airway smooth muscle by decreasing MLC phosphorylation by skewing the balance of RhoA/Rac1 activation towards Rac1. Therefore, activation of Epac may have therapeutical potential in the treatment of obstructive airway diseases.     

Cyclic AMP-dependent protein kinase and Epac mediate cyclic AMP responses in pancreatic acini

The pancreatic acinar cell has several phenotypic responses to cAMP agonists. At physiological concentrations of the muscarinic agonist carbachol (1 microM) or the CCK analog caerulein (100 pM), ligands that increase cytosolic Ca(2+), cAMP acts synergistically to enhance secretion. Supraphysiological concentrations of carbachol (1 mM) or caerulein (100 nM) suppress secretion and cause intracellular zymogen activation; cAMP enhances both zymogen activation and reverses the suppression of secretion. In addition to stimulating cAMP-dependent protein kinase (PKA), recent studies using cAMP analogs that lack a PKA response have shown that cAMP can also act through the cAMP-binding protein, Epac (exchange protein directly activated by cyclic AMP). The roles of PKA and Epac in cAMP responses were examined in isolated pancreatic acini. The activation of both cAMP-dependent pathways or the selective activation of Epac was found to enhance amylase secretion induced by physiological and supraphysiological concentrations of the muscarinic agonist carbachol. Similarly, activation of both PKA or the specific activation of Epac enhanced carbachol-induced activation of trypsinogen and chymotrypsinogen. Disorganization of the apical actin cytoskeleton has been linked to the decreased secretion observed with supraphysiological concentrations of carbachol and caerulein. Although stimulation of PKA and Epac or Epac alone could largely overcome the decreased secretion observed with either supraphysiological carbachol or caerulein, stimulation of cAMP pathways did not reduce the disorganization of the apical cytoskeleton. These studies demonstrate that PKA and Epac pathways are coupled to both secretion and zymogen activation in the pancreatic acinar cell.     

Identification and Validation of Modulators of Exchange Protein Activated by cAMP (Epac) Activity: STRUCTURE-FUNCTION IMPLICATIONS FOR Epac ACTIVATION AND INHIBITION*

The signaling molecule cAMP primarily mediates its effects by activating PKA and/or exchange protein activated by cAMP (Epac). Epac has been implicated in many responses in cells, but its precise roles have been difficult to define in the absence of Epac inhibitors. Epac, a guanine nucleotide exchange factor for the low molecular weight G protein Rap, is directly activated by cAMP. Using a bioluminescence resonance energy transfer-based assay (CAMYEL) to examine modulators of Epac activity, we took advantage of its intramolecular movement that occurs upon cAMP binding to assess Epac activation. We found that the use of CAMYEL can detect the binding of cAMP analogs to Epac and their modulation of its activity and can distinguish between agonists (cAMP), partial agonists (8-chlorophenylthio-cAMP), and super agonists (8-chlorophenylthio-2′-O-Me-cAMP). The CAMYEL assay can also identify competitive and uncompetitive Epac inhibitors, e.g. (R_p)-cAMPS and CE3F4, respectively. To confirm the results with the CAMYEL assay, we used Swiss 3T3 cells and assessed the ability of cyclic nucleotide analogs to modulate the activity of Epac or PKA, determined by Rap1 activity or VASP phosphorylation, respectively. We used computational molecular modeling to analyze the interaction of analogs with Epac1. The results reveal a rapid means to identify modulators (potentially including allosteric inhibitors) of Epac activity that also provides insight into the mechanisms of Epac activation and inhibition.