Search

AimEpac1-/- mice, but not Epac2-/- mice have elevated baseline permeability to albumin. This study extends the investigations of how Epac-dependent pathways modulate transvascular exchange in response to the classical inflammatory agent histamine. It also evaluates the limitations of models of blood-to-tissue exchange in transgenic mice in DCE-MRI measurements.MethodsWe measured DCE-MRI signal intensity in masseter muscle of wt and Epac1-/- mice with established approaches from capillary physiology to determine how changes in blood flow and vascular permeability contribute to overall changes of microvascular flux. We used two tracers, the high molecular weight tracer (Gadomer-17, MW 17 kDa, apparent MW 30-35 kDa) is expected to be primarily limited by diffusion and therefore less dependent on changes in blood flow and the low molecular weight tracer (Dotarem (MW 0.56 kDa) whose transvascular exchange is determined by both blood flow and permeability. Paired experiments in each animal combined with analytical methods provided an internally consistent description of microvascular transport.ResultsEpac1-/- mice had elevated baseline permeability relative to wt control mice for Dotarem and Gadomer-17. In contrast to wt mice, Epac1-/- mice failed to increase transvascular permeability in response to histamine. Dotarem underestimated blood flow and vascular volume and Gadomer-17 has limited sensitivity in extravascular accumulation.ConclusionThe study suggests that the normal barrier loosening effect of histamine in venular microvessels do not function when the normal barrier tightening effect of Epac1 is already compromised. The study also demonstrated that the numerical analysis of DCE-MRI data with tracers of different molecular weight has significant limitations.

AimEpac1-/- mice, but not Epac2-/- mice have elevated baseline permeability to albumin. This study extends the investigations of how Epac-dependent pathways modulate transvascular exchange in response to the classical inflammatory agent histamine. It also evaluates the limitations of models of blood-to-tissue exchange in transgenic mice in DCE-MRI measurements.MethodsWe measured DCE-MRI signal intensity in masseter muscle of wt and Epac1-/- mice with established approaches from capillary physiology to determine how changes in blood flow and vascular permeability contribute to overall changes of microvascular flux. We used two tracers, the high molecular weight tracer (Gadomer-17, MW 17 kDa, apparent MW 30-35 kDa) is expected to be primarily limited by diffusion and therefore less dependent on changes in blood flow and the low molecular weight tracer (Dotarem (MW 0.56 kDa) whose transvascular exchange is determined by both blood flow and permeability. Paired experiments in each animal combined with analytical methods provided an internally consistent description of microvascular transport.ResultsEpac1-/- mice had elevated baseline permeability relative to wt control mice for Dotarem and Gadomer-17. In contrast to wt mice, Epac1-/- mice failed to increase transvascular permeability in response to histamine. Dotarem underestimated blood flow and vascular volume and Gadomer-17 has limited sensitivity in extravascular accumulation.ConclusionThe study suggests that the normal barrier loosening effect of histamine in venular microvessels do not function when the normal barrier tightening effect of Epac1 is already compromised. The study also demonstrated that the numerical analysis of DCE-MRI data with tracers of different molecular weight has significant limitations.

Exchange proteins directly activated by cAMP (Epac1 and Epac2) have been recently recognized as key players in β-adrenergic-dependent cardiac arrhythmias. Whereas Epac1 overexpression can lead to cardiac hypertrophy and Epac2 activation can be arrhythmogenic, it is unknown whether distinct subcellular distribution of Epac1 vs. Epac2 contributes to differential functional effects. Here, we characterized and used a novel fluorescent cAMP derivate Epac ligand 8-[Pharos-575]-2'-O-methyladenosine-3',5'-cyclic monophosphate (Φ-O-Me-cAMP) in mice lacking either one or both isoforms (Epac1-KO, Epac2-KO, or double knockout, DKO) to assess isoform localization and function. Fluorescence of Φ-O-Me-cAMP was enhanced by binding to Epac. Unlike several Epac-specific antibodies tested, Φ-O-Me-cAMP exhibited dramatically reduced signals in DKO myocytes. In WT, the apparent binding affinity (Kd = 10.2 ± 0.8 µM) is comparable to that of cAMP and nonfluorescent Epac-selective agonist 8-(4-chlorophenylthio)-2-O-methyladenosine-3'-,5'-cyclicmonophosphate (OMe-CPT). Φ-O-Me-cAMP readily entered intact myocytes, but did not activate PKA and its binding was competitively inhibited by OMe-CPT, confirming its Epac specificity. Φ-O-Me-cAMP is a weak partial agonist for purified Epac, but functioned as an antagonist for four Epac signaling pathways in myocytes. Epac2 and Epac1 were differentially concentrated along T tubules and around the nucleus, respectively. Epac1-KO abolished OMe-CPT-induced nuclear CaMKII activation and export of transcriptional regulator histone deacetylase 5. In conclusion, Epac1 is localized and functionally involved in nuclear signaling, whereas Epac2 is located at the T tubules and regulates arrhythmogenic sarcoplasmic reticulum Ca leak.

Exchange proteins directly activated by cAMP (Epac1 and Epac2) have been recently recognized as key players in β-adrenergic-dependent cardiac arrhythmias. Whereas Epac1 overexpression can lead to cardiac hypertrophy and Epac2 activation can be arrhythmogenic, it is unknown whether distinct subcellular distribution of Epac1 vs. Epac2 contributes to differential functional effects. Here, we characterized and used a novel fluorescent cAMP derivate Epac ligand 8-[Pharos-575]-2'-O-methyladenosine-3',5'-cyclic monophosphate (Φ-O-Me-cAMP) in mice lacking either one or both isoforms (Epac1-KO, Epac2-KO, or double knockout, DKO) to assess isoform localization and function. Fluorescence of Φ-O-Me-cAMP was enhanced by binding to Epac. Unlike several Epac-specific antibodies tested, Φ-O-Me-cAMP exhibited dramatically reduced signals in DKO myocytes. In WT, the apparent binding affinity (Kd = 10.2 ± 0.8 µM) is comparable to that of cAMP and nonfluorescent Epac-selective agonist 8-(4-chlorophenylthio)-2-O-methyladenosine-3'-,5'-cyclicmonophosphate (OMe-CPT). Φ-O-Me-cAMP readily entered intact myocytes, but did not activate PKA and its binding was competitively inhibited by OMe-CPT, confirming its Epac specificity. Φ-O-Me-cAMP is a weak partial agonist for purified Epac, but functioned as an antagonist for four Epac signaling pathways in myocytes. Epac2 and Epac1 were differentially concentrated along T tubules and around the nucleus, respectively. Epac1-KO abolished OMe-CPT-induced nuclear CaMKII activation and export of transcriptional regulator histone deacetylase 5. In conclusion, Epac1 is localized and functionally involved in nuclear signaling, whereas Epac2 is located at the T tubules and regulates arrhythmogenic sarcoplasmic reticulum Ca leak.

The cAMP signalling pathway plays an essential role in immune functions. In the present study we examined the role of the cAMP/EPAC1 (exchange protein directly activated by cAMP) axis in regulatory T-cell (Treg)-mediated immunosuppression using genetic and pharmacological approaches. Genetic deletion of EPAC1 in Tregs and effector T-cells (Teffs) synergistically attenuated Treg-mediated suppression of Teffs. Mechanistically, EPAC1 inhibition enhanced activation of the transcription factor STAT3 (signal transducer and activator of transcription 3) and up-regulated SMAD7 expression while down-regulating expression of SMAD4. Consequently, CD4+ T-cells were desensitized to transforming growth factor (TGF) β1, a cytokine employed by Tregs to exert a broad inhibitory function within the immune system. Furthermore, deletion of EPAC1 led to production of significant levels of ovalbumin IgG antibodies in a low-dose, oral-tolerance mouse model. These in vivo observations are consistent with the finding that EPAC1 plays an important role in Treg-mediated suppression. More importantly, pharmacological inhibition of EPAC1 using an EPAC-specific inhibitor recapitulates the EPAC1 deletion phenotype both in vivo and in vitro. The results of the present study show that EPAC1 boosts Treg-mediated suppression, and identifies EPAC1 as a target with broad therapeutic potential because Tregs are involved in numerous pathologies, including autoimmunity, infections and a wide range of cancers.

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. (Rp)-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.

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. (Rp)-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.