Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP as a stimulus for Ca2+-induced Ca2+ release and exocytosis in pancreatic beta-cells

The second messenger cAMP exerts powerful stimulatory effects on Ca(2+) signaling and insulin secretion in pancreatic beta-cells. Previous studies of beta-cells focused on protein kinase A (PKA) as a downstream effector of cAMP action. However, it is now apparent that cAMP also exerts its effects by binding to cAMP-regulated guanine nucleotide exchange factors (Epac). Although one effector of Epac is the Ras-related G protein Rap1, it is not fully understood what the functional consequences of Epac-mediated signal transduction are at the cellular level. 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate (8-pCPT-2'-O-Me-cAMP) is a newly described cAMP analog, and it activates Epac but not PKA. Here we demonstrate that 8-pCPT-2'-O-Me-cAMP acts in human pancreatic beta-cells and INS-1 insulin-secreting cells to mobilize Ca(2+) from intracellular Ca(2+) stores via Epac-mediated Ca(2+)-induced Ca(2+) release (CICR). The cAMP-dependent increase of [Ca(2+)](i) that accompanies CICR is shown to be coupled to exocytosis. We propose that the interaction of cAMP and Epac to trigger CICR explains, at least in part, the blood glucose-lowering properties of an insulinotropic hormone (glucagon-like peptide-1, also known as GLP-1) now under investigation for use in the treatment of type-2 diabetes mellitus.     

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.     

Epac-selective cAMP analogs: new tools with which to evaluate the signal transduction properties of cAMP-regulated guanine nucleotide exchange factors

The identification of 2'-O-methyl substituted adenosine-3',5'-cyclic monophosphate (cAMP) analogs that activate the Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs, also known as Epac1 and Epac2), has ushered in a new era of cyclic nucleotide research in which previously unrecognized signalling properties of the second messenger cAMP have been revealed. These Epac-Selective Cyclic AMP Analogs (ESCAs) incorporate a 2'-O-methyl substitution on the ribose ring of cAMP, a modification that impairs their ability to activate protein kinase A (PKA), while leaving intact their ability to activate Epac (the Exchange Protein directly Activated by Cyclic AMP). One such ESCA in wide-spread use is 8-pCPT-2'-O-Me-cAMP. It is a cell-permeant derivative of 2'-O-Me-cAMP, and it is a super activator of Epac. A wealth of newly published studies demonstrate that 8-pCPT-2'-O-Me-cAMP is a unique tool with which to asses atypical actions of cAMP that are PKA-independent. Particularly intriguing are recent reports demonstrating that ESCAs reproduce the PKA-independent actions of ligands known to stimulate Class I (Family A) and Class II (Family B) GTP-binding protein-coupled receptors (GPCRs). This topical review summarizes the current state of knowledge regarding the molecular pharmacology and signal transduction properties of Epac-selective cAMP analogs. Special attention is focused on the rational drug design of ESCAs in order to improve their Epac selectivity, membrane permeability, and stability. Also emphasized is the usefulness of ESCAs as new tools with which to assess the role of Epac as a determinant of intracellular Ca2+ signalling, ion channel function, neurotransmitter release, and hormone secretion.     

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.     

ACTH inhibits bTREK-1 K+ channels through multiple cAMP-dependent signaling pathways

Bovine adrenal zona fasciculata (AZF) cells express bTREK-1 K(+) channels that set the resting membrane potential and function pivotally in the physiology of cortisol secretion. Inhibition of these K(+) channels by adrenocorticotropic hormone (ACTH) or cAMP is coupled to depolarization and Ca(2+) entry. The mechanism of ACTH and cAMP-mediated inhibition of bTREK-1 was explored in whole cell patch clamp recordings from AZF cells. Inhibition of bTREK-1 by ACTH and forskolin was not affected by the addition of both H-89 and PKI (6-22) amide to the pipette solution at concentrations that completely blocked activation of cAMP-dependent protein kinase (PKA) in these cells. The ACTH derivative, O-nitrophenyl, sulfenyl-adrenocorticotropin (NPS-ACTH), at concentrations that produced little or no activation of PKA, inhibited bTREK-1 by a Ca(2+)-independent mechanism. Northern blot analysis showed that bovine AZF cells robustly express mRNA for Epac2, a guanine nucleotide exchange protein activated by cAMP. The selective Epac activator, 8-pCPT-2'-O-Me-cAMP, applied intracellularly through the patch pipette, inhibited bTREK-1 (IC(50) = 0.63 microM) at concentrations that did not activate PKA. Inhibition by this agent was unaffected by PKA inhibitors, including RpcAMPS, but was eliminated in the absence of hydrolyzable ATP. Culturing AZF cells in the presence of ACTH markedly reduced the expression of Epac2 mRNA. 8-pCPT-2'-O-Me-cAMP failed to inhibit bTREK-1 current in AZF cells that had been treated with ACTH for 3-4 d while inhibition by 8-br-cAMP was not affected. 8-pCPT-2'-O-Me-cAMP failed to inhibit bTREK-1 expressed in HEK293 cells, which express little or no Epac2. These findings demonstrate that, in addition to the well-described PKA-dependent TREK-1 inhibition, ACTH, NPS-ACTH, forskolin, and 8-pCPT-2'-O-Me-cAMP also inhibit these K(+) channels by a PKA-independent signaling pathway. The convergent inhibition of bTREK-1 through parallel PKA- and Epac-dependent mechanisms may provide for failsafe membrane depolarization by ACTH.     

Structural dynamics in the activation of Epac

Epac1 is a cAMP-responsive exchange factor for the small G-protein Rap. It consists of a regulatory region containing a cyclic nucleotide binding (CNB) domain and a catalytic region that activates Rap. In the absence of cAMP, access of Rap to the catalytic site is blocked by the regulatory region. We analyzed the conformational states of the CNB domain in the absence and in the presence of cAMP and cAMP analogues by NMR spectroscopy, resulting in the first direct insights into the activation mechanism of Epac. We prove that the CNB domain exists in equilibrium between the inactive and the active conformation, which is shifted by binding of cAMP. cAMP binding results in conformational changes in both the ligand binding pocket and the outer helical segments. We used two different cAMP antagonists that block these successive changes to elucidate the steps of this process. Highlighting the role of dynamics, the superactivator 8-pCPT-2'-O-Me-cAMP induces similar conformational changes as cAMP but causes different internal mobility. The results reveal the critical elements of the CNB domain of Epac required for activation and highlight the role of dynamics in this process.     

The cAMP-responsive Rap1 guanine nucleotide exchange factor, Epac, induces smooth muscle relaxation by down-regulation of RhoA activity

Agonist activation of the small GTPase, RhoA, and its effector Rho kinase leads to down-regulation of smooth muscle (SM) myosin light chain phosphatase activity, an increase in myosin light chain (RLC(20)) phosphorylation and force. Cyclic nucleotides can reverse this process. We report a new mechanism of cAMP-mediated relaxation through Epac, a GTP exchange factor for the small GTPase Rap1 resulting in an increase in Rap1 activity and suppression of RhoA activity. An Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP ("007"), significantly reduced agonist-induced contractile force, RLC(20), and myosin light chain phosphatase phosphorylation in both intact and permeabilized vascular, gut, and airway SMs independently of PKA and PKG. The vasodilator PGI(2) analog, cicaprost, increased Rap1 activity and decreased RhoA activity in intact SMs. Forskolin, phosphodiesterase inhibitor isobutylmethylxanthine, and isoproterenol also significantly increased Rap1-GTP in rat aortic SM cells. The PKA inhibitor H89 was without effect on the 007-induced increase in Rap1-GTP. Lysophosphatidic acid-induced RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts, consistent with Epac signaling through Rap1B to down-regulate RhoA activity. Isoproterenol-induced increase in Rap1 activity was inhibited by silencing Epac1 in rat aortic SM cells. Evidence is presented that cooperative cAMP activation of PKA and Epac contribute to relaxation of SM. Our findings demonstrate a cAMP-mediated signaling mechanism whereby activation of Epac results in a PKA-independent, Rap1-dependent Ca(2+) desensitization of force in SM through down-regulation of RhoA activity. Cyclic AMP inhibition of RhoA is mediated through activation of both Epac and PKA.     

Potentiating role of cyclic AMP in pancreatic enzyme secretion, demonstrated by means of forskolin

The role of cyclic AMP in the regulation of enzyme secretion by the rabbit pancreas has been investigated by means of forskolin, an activator of the catalytic subunit of adenylate cyclase. Forskolin increases the cyclic AMP level in isolated pancreatic acini in a dose-dependent way. Basal amylase release, however, remains unchanged. Forskolin potentiates the increase in amylase release induced by the C-terminal octapeptide of cholecystokinin (CCK-8). Potentiation is already apparent at hormone concentrations which are only marginally effective in stimulating amylase secretion. CCK-8 alone does not raise the cellular cAMP level, but it potentiates the forskolin-induced increase. In relative terms, potentiation is higher with decreasing concentration of forskolin. These results indicate that cAMP alone does not play a direct role in CCK-stimulated pancreatic enzyme secretion in the rabbit, but it potentiates enzyme secretion already stimulated through a cAMP-independent process.     

Regulation of protein phosphorylation in pancreatic acini by cyclic AMP-mediated secretagogues: interaction with carbamylcholine

The effects on protein phosphorylation in mouse pancreatic acini of cyclic AMP-mediated secretagogues and the Ca2+-mediated agonist carbamylcholine were compared. Under the conditions adopted for the study of protein phosphorylation, carbamylcholine (3 microM) stimulated amylase release from pancreatic acini 6-fold, whereas vasoactive intestinal polypeptide (VIP) (100 nM) and the cyclic AMP analogue 8-bromo-cyclic AMP (1 mM) caused little or no increase in secretion. However, VIP and 8-bromo-cyclic AMP, when added in combination with carbamylcholine, potentiated the stimulation of amylase release to 170-180% of that caused by carbamylcholine alone. As assessed by two-dimensional gel electrophoresis, VIP reproduced four of the ten changes in protein phosphorylation elicited by carbamylcholine, these changes being the increased phosphorylation of one soluble protein and the decreased phosphorylation of three soluble proteins. VIP enhanced the carbamylcholine-induced changes in phosphorylation for three proteins. In addition, VIP increased the phosphorylation of a unique protein of Mr 52,000 and pI 5.66 which was not affected by carbamylcholine. All of the effects on protein phosphorylation exerted by VIP in the presence or absence of carbamylcholine were mimicked by 8-bromo-cyclic AMP. Secretin also reproduced most of the changes in protein phosphorylation caused by VIP, although concentrations of secretin of at least 100-fold higher were required to elicit a maximal response. It is concluded that cyclic AMP-mediated secretagogues alter the phosphorylation of a unique protein as well as of several pancreatic proteins affected by carbamylcholine. Moreover, these effects appear to be mediated primarily by VIP-preferring receptors and may be involved in the synergistic action of VIP to promote carbamylcholine-induced amylase release.     

Effects of increased intracellular cAMP on carbachol-stimulated zymogen activation, secretion, and injury in the pancreatic acinar cell

A characteristic of acute pancreatitis is the premature activation and retention of enzymes within the pancreatic acinar cell. Because ligands linked to cAMP production may prevent some forms of pancreatitis, we evaluated the effects of increased intracellular cAMP in the rat pancreatic acinar cell. Specifically, this study examined the effects of the cholinergic agonist carbachol and agents that increase cAMP [secretin and 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP)] on zymogen activation (trypsin and chymotrypsin), enzyme secretion, and cellular injury in isolated pancreatic acini. Although cAMP agonists affected the responses to physiological concentrations of carbachol (1 microM), their most prominent effects were observed with supraphysiological concentrations (1 mM). When secretin was added to 1 mM carbachol, there was a slight increase in zymogen activation, but no change in the secretion of amylase or chymotrypsin. Furthermore, coaddition of secretin increased parameters of cell injury (trypan blue exclusion, lactic dehydrogenase release, and morphological markers) compared with carbachol (1 mM) alone. Although directly increasing cellular cAMP by 8-Br-cAMP caused much greater zymogen activation than carbachol (1 mM) alone or with secretin, 8-Br-cAMP cotreatment reduced all parameters of injury to the level of unstimulated acini. Furthermore, 8-Br-cAMP dramatically enhanced the secretion of amylase and chymotrypsin from the acinar cell. This study demonstrates that increasing acinar cell cAMP can overcome the inhibition of enzyme secretion caused by high concentrations of carbachol and eliminate acinar cell injury.     

Phosphorylation of 3 particulate proteins in rat pancreatic acini in response to vasoactive intestinal peptide (VIP), secretin and cholecystokinin (CCK-8)

Rat pancreatic acini were preincubated with 0.4 mM 32Pi for 45 min at 37 degrees C, then exposed for 15 min to VIP, secretin or CCK-8. The incubation was terminated with a stop solution and a fraction rich in mitochondria and zymogen granules was separated from a microsome-rich fraction by differential centrifugation. After heating in the presence of SDS, beta-mercaptoethanol was added and the pattern of equivalent amounts of 32P-labelled proteins was examined by autoradiography of SDS-PAGE gels. VIP, secretin, and CCK-8 stimulated the phosphorylation of a Mr=33 K microsomal protein and that of two proteins of Mr=21 K and Mr=25 K mostly present in a fraction rich in mitochondria and zymogen granules. Stimulations were dose-dependent, the highest stimulant concentrations tested allowing 2- to 3-fold increases of phosphorylation over basal. When 1 nM CCK-8 was used simultaneously with 1 microM VIP, the cyclic AMP levels attained and the pattern of protein phosphorylation were similar to those obtained with VIP alone, and there was a potentiation of amylase secretion; when a supra-maximal 0.1 microM CCK-8 concentration was added, the VIP-induced elevation in cyclic AMP levels and the phosphorylation of the Mr=21 K and Mr=25 K proteins were partially antagonized, and no potentiation any more of secretion occurred. To conclude the in vitro phosphorylation of three particulate proteins (Mr=33 K, 25 K, and 21 K) was similarly increased in rat pancreatic acini in response to secretin and VIP (acting through cyclic AMP) and to CCK-8 (acting mostly through Ca2+).     

Cyclic AMP-Rap1A signaling activates RhoA to induce α2c-adrenoceptor translocation to the cell surface of microvascular smooth muscle cells

Intracellular signaling by the second messenger cyclic AMP (cAMP) activates the Ras-related small GTPase Rap1 through the guanine exchange factor Epac. This activation leads to effector protein interactions, activation, and biological responses in the vasculature, including vasorelaxation. In vascular smooth muscle cells derived from human dermal arterioles (microVSM), Rap1 selectively regulates expression of G protein-coupled α(2C)-adrenoceptors (α(2C)-ARs) through JNK-c-jun nuclear signaling. The α(2C)-ARs are generally retained in the trans-Golgi compartment and mobilize to the cell surface and elicit vasoconstriction in response to cellular stress. The present study used human microVSM to examine the role of Rap1 in receptor localization. Complementary approaches included murine microVSM derived from tail arteries of C57BL6 mice that express functional α(2C)-ARs and mice deficient in Rap1A (Rap1A-null). In human microVSM, increasing intracellular cAMP by direct activation of adenylyl cyclase by forskolin (10 μM) or selectively activating Epac-Rap signaling by the cAMP analog 8-pCPT-2'-O-Me-cAMP (100 μM) activated RhoA, increased α(2C)-AR expression, and reorganized the actin cytoskeleton, increasing F-actin. The α(2C)-ARs mobilized from the perinuclear region to intracellular filamentous structures and to the plasma membrane. Similar results were obtained in murine wild-type microVSM, coupling Rap1-Rho-actin dynamics to receptor relocalization. This signaling was impaired in Rap1A-null murine microVSM and was rescued by delivery of constitutively active (CA) mutant of Rap1A. When tested in heterologous HEK293 cells, Rap1A-CA or Rho-kinase (ROCK-CA) caused translocation of functional α(2C)-ARs to the cell surface (～4- to 6-fold increase, respectively). Together, these studies support vascular bed-specific physiological role of Rap1 and suggest a role in vasoconstriction in microVSM.     

Evidence against cyclic GMP as a mediator of the actions of secretagogues on amylase release from guinea-pig pancreas

In dispersed acini from guinea-pig pancrease several pancreatic secretagogues increased calcium outflux, cyclic GMP and amylase secretion, whereas nitroprusside and hydroxylamide increased cyclic GMP but did not increase calcium outflux or amylase secretion and did not alter the action of secretagogues on calcium outflux or amylase secretion. Secretin and vasoactive intestinal peptide increased cyclic AMP and increased secretion but did not alter cyclic GMP. Nitroprusside and hydroxylamine did not alter cyclic AMP or the action of secretin or vasoactive intestinal peptide on cyclic AMP and enzyme secretion. Agents that increased cyclic GMP also caused release of the nucleotide into the extracellular medium; however, this release did not correlate with secretion of amylase into the extracellular medium. 8-Bromo cyclic AMP as well as 8-bromo cyclic GMP increased enzyme secretion and potentiated the increase in enzyme secretion caused by cholecystokinin or carbachol. The increase in amylase secretion caused by vasoactive intestinal peptide or secretin plus either of the cyclic nucleotide derivatives was the same as that caused by the peptide alone. These results indicate that cyclic GMP does not mediate the action of secretagogues on pancreatic enzyme secretion, that the release of cyclic GMP into the extracellular medium does not occur by exocytosis and that the increase in enzyme secretion caused by 8-bromo cyclic GMP results from its stability to mimic the action of endogenous cyclic AMP.     

The cAMP signaling system inhibits the repair of γ-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells

Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on γ-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (GαsQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of GαsQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after γ-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2'-O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2'-O-Me-cAMP and restored XRCC1 protein level following γ-ray irradiation. From these results, we conclude that the cAMP signaling system inhibits the repair of γ-ray-induced DNA damage by promoting the ubiquitin-proteasome dependent degradation of XRCC1 in an Epac-dependent pathway in lung cancer cells.     

Role of Epac1, an exchange factor for Rap GTPases, in endothelial microtubule dynamics and barrier function

Rap1 GTPase activation by its cAMP responsive nucleotide exchange factor Epac present in endothelial cells increases endothelial cell barrier function with an associated increase in cortical actin. Here, Epac1 was shown to be responsible for these actin changes and to colocalize with microtubules in human umbilical vein endothelial cells. Importantly, Epac activation with a cAMP analogue, 8-pCPT-2'O-Me-cAMP resulted in a net increase in the length of microtubules. This did not require cell-cell interactions or Rap GTPase activation, and it was attributed to microtubule growth as assessed by time-lapse microscopy of human umbilical vein endothelial cell expressing fluorophore-linked microtubule plus-end marker end-binding protein 3. An intact microtubule network was required for Epac-mediated changes in cortical actin and barrier enhancement, but it was not required for Rap activation. Finally, Epac activation reversed microtubule-dependent increases in vascular permeability induced by tumor necrosis factor-alpha and transforming growth factor-beta. Thus, Epac can directly promote microtubule growth in endothelial cells. This, together with Rap activation leads to an increase in cortical actin, which has functional significance for vascular permeability.     

The effects of γ-hexachlorocyclohexane on amylase secretion and inositol phospholipid metabolism in mouse pancreatic acini

Dispersed mouse and guinea-pig pancreatic acini were used to examine the effects of the inositol analogue, γ-hexachlorocyclohexane (lindane) on agonist-stimulated amylase secretion. Secretion from mouse acini in response to carbachol and cholecystokinin octapeptide (CCK-8) was reduced by lindane. Similarly, amylase release from guinea-pig acini stimulated by carbachol was abolished by lindane. These acini, however, still remained responsive to dibutyryl-cAMP with only a slightly diminished secretion to this agent. Inositol phospholipid synthesis and hydrolysis was stimulated in mouse acini by both carbachol and CCK-8. Although hydrolysis of these lipids in response to CCK-8 was reduced by only 18%, stimulation of inositol phospholipid synthesis by either agonist was abolished by lindane. Dose-response curves for inositol phospholipid synthesis stimulated by carbachol and CCK-8 in mouse acini were biphasic and superimposable with those of amylase secretion. In contrast, the dose-response curve for phosphoinositide hydrolysis was sigmoid and clearly separable from that of synthesis. Reducing the external Ca²⁺ concentration caused the dose-response curves for carbachol- and CCK-8-induced inositol phospholipid synthesis to be displaced to the right, as has been observed for amylase secretion. A23187 was also found to induce amylase secretion and inositol phospholipid synthesis, and both of these responses were inhibited by lindane. Amylase secretion and inositol phospholipid synthesis may, therefore, be closely related events in the exocrine pancreas. Lindane may provide a valuable tool with which to determine the role of inositol phospholipid metabolism in stimulus-response coupling.     

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.     

A Novel Interplay between Rap1 and PKA Regulates Induction of Angiogenesis in Prostate Cancer

Angiogenesis inhibition is an important therapeutic strategy for advanced stage prostate cancer. Previous work from our laboratory showed that sustained stimulation of Rap1 by 8-pCPT-2''-O-Me-cAMP (8CPT) via activation of Epac, a Rap1 GEF, or by expression of a constitutively active Rap1 mutant (cRap1) suppresses endothelial cell chemotaxis and subsequent angiogenesis. When we tested this model in the context of a prostate tumor xenograft, we found that 8CPT had no significant effect on prostate tumor growth alone. However, in cells harboring cRap1, 8CPT dramatically inhibited not only prostate tumor growth but also VEGF expression and angiogenesis within the tumor microenvironment. Subsequent analysis of the mechanism revealed that, in prostate tumor epithelial cells, 8CPT acted via stimulation of PKA rather than Epac/Rap1. PKA antagonizes Rap1 and hypoxic induction of 1α protein expression, VEGF production and, ultimately, angiogenesis. Together these findings provide evidence for a novel interplay between Rap1, Epac, and PKA that regulates tumor-stromal induction of angiogenesis.