Exchange Protein Directly Activated By cAMP Activation Research Articles

Absinthin, an agonist of the bitter taste receptor hTAS2R46, uncovers an ER-to-mitochondria Ca2+–shuttling event

Type 2 taste receptors (TAS2R) are G protein-coupled receptors first described in the gustatory system, but have also been shown to have extraoral localizations, including airway smooth muscle (ASM) cells, in which TAS2R have been reported to induce relaxation. TAS2R46 is an unexplored subtype that responds to its highly specific agonist absinthin. Here, we first demonstrate that, unlike other bitter-taste receptor agonists, absinthin alone (1 μm) in ASM cells does not induce Ca2+ signals but reduces histamine-induced cytosolic Ca2+ increases. To investigate this mechanism, we introduced into ASM cells aequorin-based Ca2+ probes targeted to the cytosol, subplasma membrane domain, or the mitochondrial matrix. We show that absinthin reduces cytosolic histamine-induced Ca2+ rises and simultaneously increases Ca2+ influx into mitochondria. We found that this effect is inhibited by the potent human TAS2R46 (hTAS2R46) antagonist 3β-hydroxydihydrocostunolide and is no longer evident in hTAS2R46-silenced ASM cells, indicating that it is hTAS2R46-dependent. Furthermore, these changes were sensitive to the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone (FCCP); the mitochondrial calcium uniporter inhibitor KB-R7943 (carbamimidothioic acid); the cytoskeletal disrupter latrunculin; and an inhibitor of the exchange protein directly activated by cAMP (EPAC), ESI-09. Similarly, the β2 agonist salbutamol also could induce Ca2+ shuttling from cytoplasm to mitochondria, suggesting that this new mechanism might be generalizable. Moreover, forskolin and an EPAC activator mimicked this effect in HeLa cells. Our findings support the hypothesis that plasma membrane receptors can positively regulate mitochondrial Ca2+ uptake, adding a further facet to the ability of cells to encode complex Ca2+ signals.

Cyclic AMP-dependent protein kinase A and EPAC mediate VIP and secretin stimulation of PAK4 and activation of Na+,K+-ATPase in pancreatic acinar cells.

Rat pancreatic acinar cells possess only the p21-activated kinase (PAKs), PAK4 of the group II PAK, and it is activated by gastrointestinal hormones/neurotransmitters stimulating PLC and by a number of growth factors. However, little is known generally of cAMP agents causing PAK4 activation, and there are no studies with gastrointestinal hormones/neurotransmitters activating cAMP cascades. In the present study, we examined the ability of VIP and secretin, which stimulate cAMP generation in pancreatic acini, to stimulate PAK4 activation, the signaling cascades involved, and their possible role in activating sodium-potassium adenosine triphosphatase (Na+,K+-ATPase). PAK4 activation was compared with activation of the well-established cAMP target, cyclic AMP response element binding protein (CREB). Secretin-stimulated PAK4 activation was inhibited by KT-5720 and PKA Type II inhibitor (PKI), protein kinase A (PKA) inhibitors, whereas VIP activation was inhibited by ESI-09 and HJC0197, exchange protein directly activated by cAMP (EPAC) inhibitors. In contrast, both VIP/secretin-stimulated phosphorylation of CREB (pCREB) via EPAC activation; however, it was inhibited by the p44/42 inhibitor PD98059 and the p38 inhibitor SB202190. The specific EPAC agonist 8-CPT-2- O-Me-cAMP as well 8-Br-cAMP and forskolin stimulated PAK4 activation. Secretin/VIP activation of Na+,K+-ATPase, was inhibited by PAK4 inhibitors (PF-3758309, LCH-7749944). These results demonstrate PAK4 is activated in pancreatic acini by stimulation of both VIP-/secretin-preferring receptors, as is CREB. However, they differ in their signaling cascades. Furthermore, PAK4 activation is needed for Na+,K+ATPase activation, which mediates pancreatic fluid secretion. These results, coupled with recent studies reporting PAKs are involved in both pancreatitis/pancreatic cancer growth/enzyme secretion, show that PAK4, similar to PAK2, likely plays an important role in both pancreatic physiological/pathological responses. NEW & NOTEWORTHY Pancreatic acini possess only the group II p21-activated kinase, PAK4, which is activated by PLC-stimulating agents/growth factors and is important in enzyme-secretion/growth/pancreatitis. Little information exists on cAMP-activating agents stimulating group II PAKs. We studied ability/effect of cyclic AMP-stimulating agents [vasoactive intestinal polypeptide (VIP), secretin] on PAK4 activity in rat pancreatic-acini. Both VIP/secretin activated PAK4/CREB, but the cAMP signaling cascades differed for EPAC, MAPK, and PKA pathways. Both hormones require PAK4 activation to stimulate sodium-potassium adenosine triphosphatase activity. This study shows PAK4 plays an important role in VIP-/secretin-stimulated pancreatic fluid secretion and suggests it plays important roles in pancreatic acinar physiological/pathophysiological responses mediated by cAMP-activating agents.

Cyclic AMP (cAMP) confers drug resistance against DNA damaging agents via PKAIA in CML cells

Cyclic adenosine monophosphate (cAMP) regulates many vital functions such as metabolism, proliferation, differentiation and death. Depending on cell types and stimulators, cAMP could either promote or attenuate cell death. cAMP signal can be transduced by protein kinase A (PKA) and/or exchange protein directly activated by cAMP (EPAC). In CML cells, cAMP may suppress their proliferation and enhance their differentiation. However, the role of cAMP on DNA damaging agent toxicity and the mechanism involved has not been studied. In this study, we studied the effect of cAMP on the sensitivity of CML cells to DNA damaging agents. We observed that forskolin (FSK) and dibutyryl-cAMP (DBcAMP) decreased cisplatin and etoposide-induced cell death in K562 cells. Moreover, PKA activator prevented K562 cells from DNA damaging agent-induced cell death while EPAC activator had no effect. Furthermore, we found that the PKA subtype, PKAIA, was involved in cAMP-attenuated resistance in K562 cells. Taken together, our results suggest that increased cAMP level confers CML cells to acquire a novel mechanism against DNA damaging agent toxicity via PKAIA. Thus, PKAIA inhibitor may be helpful in overcoming the resistance to DNA damaging agents in CML cells.

Kv7 Channel Activation Underpins EPAC-Dependent Relaxations of Rat Arteries.

To establish the role of Kv7 channels in EPAC (exchange protein directly activated by cAMP)-dependent relaxations of the rat vasculature and to investigate whether this contributes to β-adrenoceptor-mediated vasorelaxations. Isolated rat renal and mesenteric arteries (RA and MA, respectively) were used for isometric tension recording to study the relaxant effects of a specific EPAC activator and the β-adrenoceptor agonist isoproterenol in the presence of potassium channel inhibitors and cell signaling modulators. Isolated myocytes were used in proximity ligation assay studies to detect localization of signaling intermediaries with Kv7.4 before and after cell stimulation. Our studies showed that the EPAC activator (8-pCPT-2Me-cAMP-AM) produced relaxations and enhanced currents of MA and RA that were sensitive to linopirdine (Kv7 inhibitor). Linopirdine also inhibited isoproterenol-mediated relaxations in both RA and MA. In the MA, isoproterenol relaxations were sensitive to EPAC inhibition, but not protein kinase A inhibition. In contrast, isoproterenol relaxations in RA were attenuated by protein kinase A but not by EPAC inhibition. Proximity ligation assay showed a localization of Kv7.4 with A-kinase anchoring protein in both vessels in the basal state, which increased only in the RA with isoproterenol stimulation. In the MA, but not the RA, a localization of Kv7.4 with both Rap1a and Rap2 (downstream of EPAC) increased with isoproterenol stimulation. EPAC-dependent vasorelaxations occur in part via activation of Kv7 channels. This contributes to the isoproterenol-mediated relaxation in mesenteric, but not renal, arteries.

Chenodeoxycholic acid requires activation of EGFR, EPAC, and Ca2+ to stimulate CFTR-dependent Cl- secretion in human colonic T84 cells.

Bile acids are known to initiate intricate signaling events in a variety of tissues, primarily in the liver and gastrointestinal tract. Of the known bile acids, only the 7α-dihydroxy species, deoxycholic acid and chenodeoxycholic acid (CDCA), and their conjugates, activate processes that stimulate epithelial Cl- secretion. We have previously published that CDCA acts in a rapid manner to stimulate colonic ion secretion via protein kinase A (PKA)-mediated activation of the dominant Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR) (Ao M, Sarathy J, Domingue J, Alrefai WA, and Rao MC. Am J Physiol Cell Physiol 305: C447-C456, 2013); however, PKA signaling did not account for the entire CDCA response. Here we show that in human colonic T84 cells, CDCA's induction of CFTR activity, measured as changes in short-circuit current (Isc), is dependent on epidermal growth factor receptor (EGFR) activation and does not involve the bile acid receptors TGR5 or farnesoid X receptor. CDCA activation of Cl- secretion does not require Src, mitogen-activated protein kinases, or phosphoinositide 3-kinase downstream of EGFR but does require an increase in cytosolic Ca2+ In addition to PKA signaling, we found that the CDCA response requires the novel involvement of the exchange protein directly activated by cAMP (EPAC). EPAC is a known hub for cAMP and Ca2+ cross talk. Downstream of EPAC, CDCA activates Rap2, and changes in free cytosolic Ca2+ were dependent on both EPAC and EGFR activation. This study establishes the complexity of CDCA signaling in the colonic epithelium and shows the contribution of EGFR, EPAC, and Ca2+ in CDCA-induced activation of CFTR-dependent Cl- secretion.

Proarrhythmic effect of sustained EPAC activation on TRPC3/4 in rat ventricular cardiomyocytes

The Exchange Protein directly Activated by cAMP (EPAC) participates to the pathological signaling of cardiac hypertrophy and heart failure, in which the role of Ca2+ entry through the Transient Receptor Potential Canonical (TRPC) channels begin to be appreciated. Here we studied whether EPAC activation could influence the activity and/or expression of TRPC channels in cardiac myocytes. In adult rat ventricular myocytes treated for 4 to 6h with the selective EPAC activator, 8-pCPT (10μM), we observed by Fluo-3 confocal fluorescence a Store-Operated Ca2+ Entry (SOCE) like-activity, which was blunted by co-incubation with EPAC inhibitors (ESI-05 and CE3F4 at 10μM). This SOCE-like activity, which was very small in control incubated cells, was sensitive to 30-μM SKF-96365. Molecular screening showed a specific upregulation of TRPC3 and C4 protein isoforms after 8-pCPT treatment. Moreover, sustained EPAC activation favored proarrhythmic Ca2+ waves, which were reduced either by co-incubation with EPAC inhibitors or bath perfusion with TRPC inhibitors.Our study provides the first evidence that sustained selective EPAC activation leads to an increase in TRPC3 and C4 protein expression and induces a proarrhythmic SOCE-like activity in adult rat ventricular cardiomyocytes, which might be of importance during the development of cardiac diseases.

EPAC Activation of Cardiac Muscle

Exchange protein-directly activated by cAMP (EPAC) has been shown to be involved in the classical cAMP-activated protein kinase A (PKA) pathway in many different cell types. However, in the cardiac muscle the effects of EPAC have yielded contradictory results with reports of increased (Oestrich, 2007) and decreased (Pereira, 2007 & Cazorla, 2009) Ca2+ transient amplitude in mice and rats, respectively. The aim of this study was to examine the role of EPAC activation in excitation-contraction (EC) coupling in isolated rat ventricular muscle preparations. To activate the EPAC pathway, we used 8-(4-chlorophenylthio)-2’-O-methyladenosine-3’, 5’-cyclic monophosphate (cpTOME), an analogue of cAMP. Simultaneous measurements of intracellular calcium ([Ca2+]i) with Fura-2 and isometric force were made in RV trabeculae before and during cpTOME application. At 1.5 mM [Ca2+]o, 10 μM cpTOME had no effect on the amplitude of [Ca2+]i transients (0.7 ± 0.1 to 0.7 ± 0.1, P = 0.35) or on twitch force (24.1 ± 5.2 mN mm−2 to 22.80 ± 4.62 mN mm−2, P = 0.20) (n = 7). However, at 0.5 mM [Ca2+]o, cpTOME increased peak stress from 10.5 ± 2.8 mN mm−2 to 15.0 ± 2.7 mN mm−2, P = 0.01 (n = 6), but without any change in [Ca2+]i transients (P = 0.16). The force-[Ca2+]i relationship of intact trabeculae exhibited increased myofilament Ca2+ sensitivity with cpTOME at low [Ca2+]o but not at 1.5 mM [Ca2+]o. In isolated cells, cpTOME increased Ca2+ spark frequency (Fluo-4) from 6.6 per 100 μm3 s (n = 3) to 32.3 per 100 μm3 s (n = 9), P = 0.05, with a reduction in the peak amplitude of the sparks. The latter result recapitulates the idea that changes in RyR sensitivity do not alter the amplitude of Ca2+ transients (Eisner, 2009).

Role of Dynamics in the Autoinhibition and Activation of the Exchange Protein Directly Activated by Cyclic AMP (EPAC)

The exchange protein directly activated by cAMP (EPAC) is a key receptor of cAMP in eukaryotes and controls critical signaling pathways. Currently, no residue resolution information is available on the full-length EPAC dynamics, which are known to be pivotal determinants of allostery. In addition, no information is presently available on the intermediates for the classical induced fit and conformational selection activation pathways. Here these questions are addressed through molecular dynamics simulations on five key states along the thermodynamic cycle for the cAMP-dependent activation of a fully functional construct of EPAC2, which includes the cAMP-binding domain and the integral catalytic region. The simulations are not only validated by the agreement with the experimental trends in cAMP-binding domain dynamics determined by NMR, but they also reveal unanticipated dynamic attributes, rationalizing previously unexplained aspects of EPAC activation and autoinhibition. Specifically, the simulations show that cAMP binding causes an extensive perturbation of dynamics in the distal catalytic region, assisting the recognition of the Rap1b substrate. In addition, analysis of the activation intermediates points to a possible hybrid mechanism of EPAC allostery incorporating elements of both the induced fit and conformational selection models. In this mechanism an entropy compensation strategy results in a low free-energy pathway of activation. Furthermore, the simulations indicate that the autoinhibitory interactions of EPAC are more dynamic than previously anticipated, leading to a revised model of autoinhibition in which dynamics fine tune the stability of the autoinhibited state, optimally sensitizing it to cAMP while avoiding constitutive activation.

Abstract 17498: PKA is the Canonical Mediator of “Fight-or-flight” Response of the Heart

β-adrenergic system is the major system regulating cardiac function under “fight-or-flight” situations. Recently it has been reported that PKA-independent mechanisms such as exchange protein directly activated by cAMP (EPAC) and PKA-independent activation of Ca/calmodulin dependent kinase II (CaMK II) also play a role in the “fight-or-flight” response (FFR). To study the relative contribution of PKA, CaMK II and EPAC to the FFR, transgenic mice (4m) with specific inhibition of PKA (PKI) and myocytes were used to determine cardiac or myocyte responses to β-adrenergic stimulation. Results: 1 . PKI was fully expressed at the age of 4 months and did not interfere with the production of cAMP induced by β-AR stimulation and the activation of CaMK II by high extracellular Ca, indicating no alteration of the components of β-AR/Gs/AC signaling pathway; 2 . ECHO showed that at baseline, the fractional shortening (FS: PKI: 38.7±1.5%; ctr: 39.0±2.1%) and ejection fraction (EF: PKI: 66.8±3.2%; ctr: 68.9.0±4.1%) were not significantly different between groups; ISO increased heart rate (HR: ctr: from 412±11bpm to 613±23bpm; PKI: from 425±13bpm to 430±21bpm), EF (ctr: 85.6±3.3%; PKI: 70.8±2.9%) and FS (ctr: 57.0±4.5%; PKI: 39.5±2.7%) in ctr but not in PKI mice. 3 . At baseline, maximal dp/dt (ctr: 6980±197mmHg/s; PKI: 7204±200mmHg/s) and minimum -dp/dt (ctr: -6248±214mmHg/s; PKI: 6587±304mmHg/s) were not different; ISO increased the HR, dp/dt (ctr: 1298±246mmHg/s; PKI: 7264±156mmHg/s) and -dp/dt (ctr: 7588±390mmHg; PKI: -5607±414mmHg) in ctr but not in TG mice. 4 . ISO increased Ca current, contraction, Ca transient and SR Ca content in ctr myocytes but not in TG myocytes. 5 . The phosphorylation of phospholamban at the PKA site (ser16) and CaMK II site (thr17) induced by ISO was blocked by PKI but not in ctr hearts, indicating that CaMK II is downstream to PKA. 6 . An EPAC activator, 8-cp-TOME-AM, could not increase cardiac performance of Langendorff perfused PKI hearts although it did slightly enhance cardiac function of ctr hearts, maybe due to its inhibitory effects on phosphodiesterases. Conclusion: PKA and its downstream CaMK II are the mediators of the “fight-or-flight” responses. Other PKA-independent pathways may not play a significant role.

Mapping allostery through the covariance analysis of NMR chemical shifts

Allostery is a fundamental mechanism of regulation in biology. The residues at the end points of long-range allosteric perturbations are commonly identified by the comparative analyses of structures and dynamics in apo and effector-bound states. However, the networks of interactions mediating the propagation of allosteric signals between the end points often remain elusive. Here we show that the covariance analysis of NMR chemical shift changes caused by a set of covalently modified analogs of the allosteric effector (i.e., agonists and antagonists) reveals extended networks of coupled residues. Unexpectedly, such networks reach not only sites subject to effector-dependent structural variations, but also regions that are controlled by dynamically driven allostery. In these regions the allosteric signal is propagated mainly by dynamic rather than structural modulations, which result in subtle but highly correlated chemical shift variations. The proposed chemical shift covariance analysis (CHESCA) identifies interresidue correlations based on the combination of agglomerative clustering (AC) and singular value decomposition (SVD). AC results in dendrograms that define functional clusters of coupled residues, while SVD generates score plots that provide a residue-specific dissection of the contributions to binding and allostery. The CHESCA approach was validated by applying it to the cAMP-binding domain of the exchange protein directly activated by cAMP (EPAC) and the CHESCA results are in full agreement with independent mutational data on EPAC activation. Overall, CHESCA is a generally applicable method that utilizes a selected chemical library of effector analogs to quantitatively decode the binding and allosteric information content embedded in chemical shift changes.

Regulation of Na+/H+exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells

The gut incretin hormone glucagon-like peptide 1 (GLP-1) is released in response to ingested nutrients and enhances insulin secretion. In addition to its insulinotropic properties, GLP-1 has been shown to have natriuretic actions paralleled by a diminished proton secretion. We therefore studied the role of the GLP-1 receptor agonist exendin-4 in modulating the activity of Na(+)/H(+) exchanger NHE3 in LLC-PK(1) cells. We found that NHE3-mediated Na(+)-dependent intracellular pH (pH(i)) recovery decreased approximately 50% after 30-min treatment with 1 nM exendin-4. Pharmacological inhibitors and cAMP analogs that selectively activate protein kinase A (PKA) or the exchange protein directly activated by cAMP (EPAC) demonstrated that regulation of NHE3 activity by exendin-4 requires activation of both cAMP downstream effectors. This conclusion was based on the following observations: 1) the PKA antagonist H-89 completely prevented the effect of the PKA activator but only partially blocked the exendin-4-induced NHE3 inhibition; 2) the MEK1/2 inhibitor U-0126 abolished the effect of the EPAC activator but only diminished the exendin-4-induced NHE3 inhibition; 3) combination of H-89 and U-0126 fully prevented the effect of exendin-4 on NHE3; 4) no additive effect in the inhibition of NHE3 activity was observed when exendin-4, PKA, and EPAC activators were used together. Mechanistically, the inhibitory effect of exendin-4 on pH(i) recovery was associated with an increase of NHE3 phosphorylation. Conversely, this inhibition took place without changes in the surface expression of the transporter. We conclude that GLP-1 receptor agonists modulate sodium homeostasis in the kidney, most likely by affecting NHE3 activity.

EPAC proteins transduce diverse cellular actions of cAMP

It has now been over 10 years since efforts to completely understand the signalling actions of cAMP (3'-5'-cyclic adenosine monophosphate) led to the discovery of exchange protein directly activated by cAMP (EPAC) proteins. In the current review we will highlight important advances in the understanding of EPAC structure and function and demonstrate that EPAC proteins mediate multiple actions of cAMP in cells, revealing future targets for pharmaceutical intervention. It has been known for some time that drugs that elevate intracellular cAMP levels have proven therapeutic benefit for diseases ranging from depression to inflammation. The challenge now is to determine which of these positive actions of cAMP involve activation of EPAC-regulated signal transduction pathways. EPACs are specific guanine nucleotide exchange factors for the Ras GTPase homologues, Rap1 and Rap2, which they activate independently of the classical routes for cAMP signalling, cyclic nucleotide-gated ion channels and protein kinase A. Rather, EPAC activation is triggered by internal conformational changes induced by direct interaction with cAMP. Leading from this has been the development of EPAC-specific agonists, which has helped to delineate numerous cellular actions of cAMP that rely on subsequent activation of EPAC. These include regulation of exocytosis and the control of cell adhesion, growth, division and differentiation. Recent work also implicates EPAC in the regulation of anti-inflammatory signalling in the vascular endothelium, namely negative regulation of pro-inflammatory cytokine signalling and positive support of barrier function. Further elucidation of these important signalling mechanisms will no doubt support the development of the next generation of anti-inflammatory drugs.

The inhibitory effect of the glucagon‐like peptide‐1 analog exendin‐4 on NHE3 activity in proximal tubule cells is mediated by both PKA and EPAC signaling pathways

Recent studies suggest a potential role for glucagon like peptide‐1 (GLP‐1) in salt and water homeostasis. Unpublished data from our laboratory indicate that exendin‐4, a GLP‐1 analog, reduces NHE3‐mediated Na/H exchange in the LLC‐PK1 cell line derived from kidney proximal tubule. The aim of this work was to define the signaling mechanisms involved in modulation of NHE3 by exendin‐4. Combination of 8‐Br‐cAMP and exendin‐4 does not result in an additive effect on the Na‐dependent intracellular pH recovery in LLC‐PK1 cells. The PKA antagonist H89 partially prevents inhibition of NHE3 activity by exendin‐4. No synergistic effect on the reduction of NHE3 activity is observed when LLC‐PK1 cells are treated simultaneously with exendin‐4 and cAMP analogs which selectively activate PKA or the exchange protein directly activated by cAMP (EPAC). Both exendin‐4 and PKA activation increase NHE3 phosphorylation levels by 70%, and this effect is completely abolished by pretreatment with H89. In contrast, EPAC activation does not alter NHE3 phosphorylation status. Collectively, these results suggest that exendin‐4 inhibition of NHE3 activity in LLC‐PK1 cells occurs through both PKA and EPAC‐dependent mechanisms.

VIP differentially activates β2 integrins, CR1, and matrix metalloproteinase-9 in human monocytes through cAMP/PKA, EPAC, and PI-3K signaling pathways via VIP receptor type 1 and FPRL1

The neuropeptide vasoactive intestinal peptide (VIP) regulates the exocytosis of secretory granules in a wide variety of cells of neuronal and non-neuronal origin. In human monocytes, we show that the proinflammatory effects of VIP are associated with stimulation of exocytosis of secretory vesicles as well as tertiary (gelatinase) granules with, respectively, up-regulation of the membrane expression of the beta2 integrin CD11b, the complement receptor 1 (CD35), and the matrix metalloproteinase-9 (MMP-9). Using the low-affinity formyl peptide receptor-like 1 (FPRL1) antagonist Trp-Arg-Trp-Trp-Trp-Trp (WRW4) and the exchange protein directly activated by cAMP (EPAC)-specific compound 8CPT-2Me-cAMP and measuring the expression of Rap1 GTPase-activating protein as an indicator of EPAC activation, we found that the proinflammatory effect of VIP is mediated via the specific G protein-coupled receptor VIP/pituitary adenylate cyclase-activating protein (VPAC1) receptor as well as via FPRL1: VIP/VPAC1 interaction is associated with a cAMP increase and activation of a cAMP/p38 MAPK pathway, which regulates MMP-9, CD35, and CD11b exocytosis, and a cAMP/EPAC/PI-3K/ERK pathway, which regulates CD11b expression; VIP/FPRL1 interaction results in cAMP-independent PI-3K/ERK activation with downstream integrin up-regulation. In FPRL1-transfected Chinese hamster ovary-K1 cells lacking VPAC1, VIP exposure also resulted in PI-3K/ERK activation. Thus, the proinflammatory effects of VIP lie behind different receptor interactions and multiple signaling pathways, including cAMP/protein kinase A, cAMP/EPAC-dependent pathways, as well as a cAMP-independent pathway, which differentially regulates p38 and ERK MAPK and exocytosis of secretory vesicles and granules.

Activation of Exchange Protein Directly Activated by Cyclic Adenosine Monophosphate and Protein Kinase A Regulate Common and Distinct Steps in Promoting Plasma Membrane Exocytic and Granule-to-Granule Fusions in Rat Islet β Cells

Using FM1-43 epifluorescence imaging and electron microscopy, we recently reported that glucagon-like peptide (GLP-1)-mediated cyclic adenosine monophosphate (cAMP) potentiation of insulin secretion markedly promotes the number of plasma membrane (PM) exocytic sites and insulin secretory granule (SG)-to-granule fusions underlying compound and sequential exocytosis. Here, we used FM1-43 imaging to dissect the distinct contributions of putative GLP-1/cAMP activated substrates--exchange protein directly activated by cAMP (EPAC) and protein kinase A (PKA)--in mediating these exocytic events. Like GLP-1, cAMP activation by forskolin increased the number of PM exocytic sites (2.3-fold), which were mainly of the robust-sustained (55.8%) and stepwise-multiphasic (37.7%) patterns corresponding to compound and sequential SG-SG exocytosis, respectively, with few monophasic hotspots (6.5%) corresponding to single-granule exocytosis. Direct activation of EPAC by 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cAMP also increased the number of exocytic sites, but which were mainly multiphasic (60%) and monophasic (40%) hotspots. Protein kinase A inhibition by H89 blocked forskolin-evoked robust-sustained hotspots, while retaining multiphasic (47%) and monophasic (53%) hotspots. Consistently, PKA activation (N6-benzoyladenosine-3',5'-cAMP) evoked only multiphasic (60%) and monophasic (40%) hotspots. These results suggested that PKA activation is required but alone is insufficient to promote compound SG-SG fusions. 8-(4-Chloro-phenylthio)-2'-O-methyladenosine-3',5'-cAMP plus N6-benzoyladenosine-3',5'-cAMP stimulation completely reconstituted the effects of forskolin, including increasing the number of exocytic sites, with a similar pattern of robust-sustained (42.6%) and stepwise (39.6%) hotspots and few monophasic (17.8%) hotspots. The EPAC and PKA modulate both distinct and common exocytic steps to potentiate insulin exocytosis where (a) EPAC activation mobilizes SGs to fuse at the PM, thereby increasing number of PM exocytic sites; and (b) PKA and EPAC activation synergistically modulate SG-SG fusions underlying compound and sequential exocytoses.

Regulation of sodium-proton exchanger isoform 3 (NHE3) by PKA and exchange protein directly activated by cAMP (EPAC)

The Na(+)/H(+) exchanger 3 (NHE3) is expressed in the brush border membrane (BBM) of proximal tubules (PT). Its activity is down-regulated on increases in intracellular cAMP levels. The aim of this study was to investigate the contribution of the protein kinase A (PKA) and the exchange protein directly activated by cAMP (EPAC) dependent pathways in the regulation of NHE3 by adenosine 3',5'-cyclic monophosphate (cAMP). Opossum kidney cells and murine kidney slices were treated with cAMP analogs, which selectively activate either PKA or EPAC. Activation of either pathway resulted in an inhibition of NHE3 activity. The EPAC-induced effect was independent of PKA as indicated by the lack of activation of the kinase and the insensitivity to the PKA inhibitor H89. Both PKA and EPAC inhibited NHE3 activity without inducing changes in the expression of the transporter in BBM. Activation of PKA, but not of EPAC, led to an increase of NHE3 phosphorylation. In contrast, activation of PKA, but not of EPAC, inhibited renal type IIa Na(+)-coupled inorganic phosphate cotransporter (NaPi-IIa), another Na-dependent transporter expressed in proximal BBM. PKA, but not EPAC, induced the retrieval of NaPi-IIa from BBM. Our results suggest that EPAC activation may represent a previously unrecognized mechanism involved in the cAMP regulation of NHE3, whereas regulation of NaPi-IIa is mediated by PKA but not by EPAC.

Microtubule-associated proteins (MAPs) regulate cAMP signalling through exchange protein directly activated by cAMP (EPAC)

cAMP is an essential signalling molecule whose concentration in cells is regulated by a wide range of hormones. A large number of diseases, including cancer and asthma, are linked to improper regulation of the cAMP signalling system, and manipulation of cAMP levels by pharmaceutical agents has proven therapeutic benefit. The action of cAMP in cells is mediated through the signalling enzymes PKA (protein kinase A) and EPAC (exchange protein directly activated by cAMP). The study of the function of these proteins is essential to understand the role of cAMP in controlling disease. We have found that EPAC interacts with an ancillary protein, called LC2 (light chain 2), and this interaction enhances EPAC's ability to activate its substrate protein, Rap1 GTPase. This is an important finding because Rap1 is involved in the control of cell migration and cell shape, functions that are disrupted in diseases like cancer. LC2 appears to enhance EPAC activity towards Rap1 by increasing the ability of EPAC to interact with cAMP, so that EPAC activation occurs at lower concentrations of cAMP. The design of inhibitors that disrupt or enhance EPAC1-LC2 interaction may therefore form the basis of future therapeutics for diseases where cAMP signalling through Rap1 is improperly regulated.

Microtubule-associated proteins (MAPs) regulate cAMP signalling through exchange protein directly activated by cAMP (EPAC).

cAMP is an essential signalling molecule whose concentration in cells is regulated by a wide range of hormones. A large number of diseases, including cancer and asthma, are linked to improper regulation of the cAMP signalling system, and manipulation of cAMP levels by pharmaceutical agents has proven therapeutic benefit. The action of cAMP in cells is mediated through the signalling enzymes PKA (protein kinase A) and EPAC (exchange protein directly activated by cAMP). The study of the function of these proteins is essential to understand the role of cAMP in controlling disease. We have found that EPAC interacts with an ancillary protein, called LC2 (light chain 2), and this interaction enhances EPAC's ability to activate its substrate protein, Rap1 GTPase. This is an important finding because Rap1 is involved in the control of cell migration and cell shape, functions that are disrupted in diseases like cancer. LC2 appears to enhance EPAC activity towards Rap1 by increasing the ability of EPAC to interact with cAMP, so that EPAC activation occurs at lower concentrations of cAMP. The design of inhibitors that disrupt or enhance EPAC1-LC2 interaction may therefore form the basis of future therapeutics for diseases where cAMP signalling through Rap1 is improperly regulated.