Synthesis and Biochemical Evaluation of Noncyclic Nucleotide Exchange Proteins Directly Activated by cAMP 1 (EPAC1) Regulators.

Exchange proteins directly activated by cAMP (EPAC) play a central role in various biological functions, and activation of the EPAC1 protein has shown potential benefits for the treatment of various human diseases. Herein, we report the synthesis and biochemical evaluation of a series of noncyclic nucleotide EPAC1 activators. Several potent EPAC1 binders were identified including 25g, 25q, 25n, 25u, 25e, and 25f, which promote EPAC1 guanine nucleotide exchange factor activity in vitro. These agonists can also activate EPAC1 protein in cells, where they exhibit excellent selectivity toward EPAC over protein kinase A and G protein-coupled receptors. Moreover, 25e, 25f, 25n, and 25u exhibited improved selectivity toward activation of EPAC1 over EPAC2 in cells. Of these, 25u was found to robustly inhibit IL-6-activated signal transducer and activator of transcription 3 (STAT3) and subsequent induction of the pro-inflammatory vascular cell adhesion molecule 1 (VCAM1) cell-adhesion protein. These novel EPAC1 activators may therefore act as useful pharmacological tools for elucidation of EPAC function and promising drug leads for the treatment of relevant human diseases.

Resveratrol improves hepatic steatosis by inducing autophagy through the cAMP signaling pathway.

SCOPE: Resveratrol (RSV), a natural polyphenol, has been reported to attenuate nonalcoholic fatty liver disease (NAFLD); however, its underlying mechanism is unclear. Autophagy was recently identified as a critical protective mechanism during NAFLD development. Therefore, we investigated the role of autophagy in the beneficial effects of RSV on hepatic steatosis. METHODS AND RESULTS: Via Oil red O staining, triglyceride, and ß-hydroxybutyrate detection, we found that RSV decreased palmitate-induced lipid accumulation and stimulated fatty acid ß-oxidation in hepatocytes. Based on Western blot assay, confocal microscopy and transmission electron microscopy, we found that RSV induced autophagy in hepatocytes, whereas autophagy inhibition markedly abolished RSV-mediated hepatic steatosis improvement. Moreover, RSV increased cAMP levels and the levels of SIRT1 (sirtuin 1), pPRKA (phosphorylated protein kinase A), and pAMPK (phosphorylated AMP-activated protein kinase), as well as SIRT1 activity in HepG2 cells. Incubation with inhibitors of AC (adenylyl cyclase), PRKA, AMPK, SIRT1, or with AC, PRKA, AMPK, or SIRT1 siRNA abolished RSV-mediated autophagy. Similar results were obtained in mice with hepatic steatosis. CONCLUSION: RSV improved hepatic steatosis partially by inducing autophagy via the cAMP-PRKA-AMPK-SIRT1 signaling pathway, which provides new evidence regarding RSV's effects on NAFLD treatment.

Interactions between hERG and KCNQ1 α-subunits are mediated by their COOH termini and modulated by cAMP.

KCNQ1 and hERG encode the voltage-gated potassium channel α-subunits of the cardiac repolarizing currents I(Ks) and I(Kr), respectively. These currents function in vivo with some redundancy to maintain appropriate action potential durations (APDs), and loss-of-function mutations in these channels manifest clinically as long QT syndrome, characterized by the prolongation of the QT interval, polymorphic ventricular tachycardia, and sudden cardiac death. Previous cellular electrophysiology experiments in transgenic rabbit cardiomyocytes and heterologous cell lines demonstrated functional downregulation of complementary repolarizing currents. Biochemical assays indicated direct, protein-protein interactions between KCNQ1 and hERG may underlie the interplay between I(Ks) and I(Kr). Our objective was to investigate hERG-KCNQ1 interactions in the intact cellular environment primarily through acceptor photobleach FRET (apFRET) experiments. We quantitatively assessed the extent of interactions based on fluorophore location and the potential regulation of interactions by physiologically relevant signals. apFRET experiments established specific hERG-KCNQ1 associations in both heterologous and primary cardiomyocytes. The largest FRET efficiency (E(f); 12.0 ± 5.2%) was seen between ion channels with GFP variants fused to the COOH termini. Acute treatment with forskolin + IBMX or a membrane-permeable cAMP analog significantly and specifically reduced the extent of hERG-KCNQ1 interactions (by 41 and 38%, respectively). Our results demonstrate direct interactions between KCNQ1 and hERG occur in both intact heterologous cells and primary cardiomyocytes and are mediated by their COOH termini. Furthermore, this interplay between channel proteins is regulated by intracellular cAMP.

The Wnt signaling pathway effector TCF7L2 controls gut and brain proglucagon gene expression and glucose homeostasis.

The type 2 diabetes risk gene TCF7L2 is the effector of the Wnt signaling pathway. We found previously that in gut endocrine L-cell lines, TCF7L2 controls transcription of the proglucagon gene (gcg), which encodes the incretin hormone glucagon-like peptide-1 (GLP-1). Whereas peripheral GLP-1 stimulates insulin secretion, brain GLP-1 controls energy homeostasis through yet-to-be defined mechanisms. We aim to determine the metabolic effect of a functional knockdown of TCF7L2 by generating transgenic mice that express dominant-negative TCF7L2 (TCF7L2DN) specifically in gcg-expressing cells. The gcg-TCF7L2DN transgenic mice showed reduced gcg expression in their gut and brain, but not in pancreas. Defects in glucose homeostasis were observed in these mice, associated with attenuated plasma insulin levels in response to glucose challenge. The defect in glucose disposal was exacerbated with high-fat diet. Brain Wnt activity and feeding-mediated hypothalamic AMP-activated protein kinase (AMPK) repression in these mice were impaired. Peripheral injection of the cAMP-promoting agent forskolin increased brain ß-cat Ser675 phosphorylation and brain gcg expression and restored feeding-mediated hypothalamic AMPK repression. We conclude that TCF7L2 and Wnt signaling control gut and brain gcg expression and glucose homeostasis and speculate that positive cross-talk between Wnt and GLP-1/cAMP signaling is an underlying mechanism for brain GLP-1 in exerting its metabolic functions.

A novel nutrient sensing mechanism underlies substrate-induced regulation of monocarboxylate transporter-1.

Monocarboxylate transporter isoform-1 (MCT1) plays an important role in the absorption of short-chain fatty acids (SCFAs) in the colon. Butyrate, a major SCFA, serves as the primary energy source for the colonic mucosa, maintains epithelial integrity, and ameliorates intestinal inflammation. Previous studies have shown substrate (butyrate)-induced upregulation of MCT1 expression and function via transcriptional mechanisms. The present studies provide evidence that short-term MCT1 regulation by substrates could be mediated via a novel nutrient sensing mechanism. Short-term regulation of MCT1 by butyrate was examined in vitro in human intestinal C2BBe1 and rat intestinal IEC-6 cells and ex vivo in rat intestinal mucosa. Effects of pectin feeding on MCT1, in vivo, were determined in rat model. Butyrate treatment (30-120 min) of C2BBe1 cells increased MCT1 function {p-(chloromercuri) benzene sulfonate (PCMBS)-sensitive [(14)C]butyrate uptake} in a pertussis toxin-sensitive manner. The effects were associated with decreased intracellular cAMP levels, increased V(max) of butyrate uptake, and GPR109A-dependent increase in apical membrane MCT1 level. Nicotinic acid, an agonist for the SCFA receptor GPR109A, also increased MCT1 function and decreased intracellular cAMP. Pectin feeding increased apical membrane MCT1 levels and nicotinate-induced transepithelial butyrate flux in rat colon. Our data provide strong evidence for substrate-induced enhancement of MCT1 surface expression and function via a novel nutrient sensing mechanism involving GPR109A as a SCFA sensor.

Activation of extracellular-signal regulated kinase by epidermal growth factor is potentiated by cAMP-elevating agents in primary cultures of adult rat hepatocytes.

We investigated the effects of α- and ß-adrenergic agonists on epidermal growth factor (EGF)-stimulated extracellular-signal regulated kinase (ERK) isoforms in primary cultures of adult rat hepatocytes. Hepatocytes were isolated and cultured with EGF (20 ng/ml) and/or α(1)-, α(2)- and ß(2)-adrenergic agonists. Phosphorylated ERK isoforms (ERK1; p44 mitogen-activated protein kinase (MAPK) and ERK2; p42 MAPK) were detected by Western blotting analysis using anti-phospho-ERK1/2 antibody. The results show that EGF induced a 2.5-fold increase in ERK2-, but not ERK1-, phosphorylation within 3 min. This EGF-induced ERK2 activation was abolished by treatment with the EGF-receptor kinase inhibitor AG1478 (10(-7) M) or the MEK (MAPK kinase) inhibitor PD98059 (10(-6) M). The α(2)-adrenergic and ß(2)-adrenergic agonists, UK14304 (10(-6) M) and metaproterenol (10(-6) M), respectively, had no effect in the absence of EGF, but metaproterenol significantly potentiated EGF-induced ERK2 phosphorylation. Moreover, the cell-permeable cAMP analog 8-bromo cAMP (10(-7) M), also potentiated EGF-induced ERK2 phosphorylation. The effects of these analogs were antagonized by the protein kinase A (PKA) inhibitor H-89 (10(-7) M). These results suggest that direct or indirect activation of PKA represents a positive regulatory mechanism for EGF stimulation of ERK2 induction.

Accelerating axon growth to overcome limitations in functional recovery after peripheral nerve injury.

OBJECTIVE: Injured peripheral nerves regenerate at very slow rates. Therefore, proximal injury sites such as the brachial plexus still present major challenges, and the outcomes of conventional treatments remain poor. This is in part attributable to a progressive decline in the Schwann cells' ability to provide a supportive milieu for the growth cone to extend and to find the appropriate target. These challenges are compounded by the often considerable delay of regeneration across the site of nerve laceration. Recently, low-frequency electrical stimulation (as brief as an hour) has shown promise, as it significantly accelerated regeneration in animal models through speeding of axon growth across the injury site. METHODS: To test whether this might be a useful clinical tool, we carried out a randomized controlled trial in patients who had experienced substantial axonal loss in the median nerve owing to severe compression in the carpal tunnel. To further elucidate the potential mechanisms, we applied rolipram, a cyclic adenosine monophosphate agonist, to rats after axotomy of the femoral nerve. RESULTS: We demonstrated that effects similar to those observed in animal studies could also be attained in humans. The mechanisms of action of electrical stimulation likely operate through up-regulation of neurotrophic factors and cyclic adenosine monophosphate. Indeed, the application of rolipram significantly accelerated nerve regeneration. CONCLUSION: With new mechanistic insights into the influencing factors of peripheral nerve regeneration, the novel treatments described above could form part of an armament of synergistic therapies that could make a meaningful difference to patients with peripheral nerve injuries.

Quantification of cAMP antagonist action in vitro and in living cells.

cAMP-dependent protein kinase (PKA) plays a key role in intracellular signalling. cAMP antagonists, acting as suppressors of PKA activity by preventing PKA-holoenzyme dissociation, have received increasing attention because of their potential use in diagnostics as well as for therapeutic purposes. A large number of cAMP analogs have been described over the last three decades and methodology has been established to monitor cAMP agonists action by either following enzymatic activity or holoenzyme dissociation. This is not the case for cAMP antagonists, where only a few substances have been demonstrated to exhibit effects in the low micromolar range, for example, Rp-8-Br-cAMPS. A main drawback in the development of new compounds is the lack of technologies to assess antagonist action in an in vitro situation as well as in living cells. Here we quantify the effect of several cAMP analogs applying three different biochemical/biophysical assay setups and one in-cell assay. This includes two methods monitoring subunit dissociation in a test tube, namely AlphaScreen, a bead-based proximity assay, and surface plasmon resonance, determining the association and dissociation patterns of the two PKA subunits in real time in response to antagonists. BRET(2), performed in living cells in a 96-well format, allows testing for the efficacy of membrane-permeable cAMP analogs based on a genetically engineered cAMP sensor. Using novel and established experimental strategies side by side, the action of cAMP and cAMP analogs was tested on type Ialpha PKA holoenzyme, thus generating methodology to screen drug libraries for potential cAMP antagonists with high accuracy, reproducibility as well as potential for automation.

Physiological concentrations of dopamine inhibit the proliferation and cytotoxicity of human CD4+ and CD8+ T cells in vitro: a receptor-mediated mechanism.

OBJECTIVE: Dopamine, a catecholamine neurotransmitter, influences growth and proliferation of lymphocytes. Pharmacological doses of dopamine have been shown to modulate T cell functions significantly, but no information is available on the effect of physiological concentrations of circulating dopamine on CD4+ and CD8+ T cell functions. This information may be of importance since significantly elevated plasma dopamine levels were observed in humans during uncoping stress, and suppression of T cell functions during stress is a well-known phenomenon. However, the mechanism inducing the suppression of T cell functions during stress is not yet clear. In the present investigation, we evaluated the effect of the dopamine level attained in the plasma of individuals with uncoping stress on the proliferation and cytotoxicity of CD4+ and CD8+ T cells in vitro. METHODS: T cell subpopulations were separated by panning. The effect of dopamine on IL-2-induced cell proliferation in vitro was evaluated by [3H]thymidine incorporation and cytotoxicity by 51Cr release, receptors by radioligand binding, cAMP by an assay kit and apoptosis by DNA fragmentation. RESULTS: At these elevated physiological concentrations, dopamine was found to inhibit significantly the proliferation and cytotoxicity of CD4+ and CD8+ T cells in vitro. This dopamine-mediated inhibition of proliferation was more marked on CD8+ T cells than on CD4+ T cells. The underlying mechanism was found to be D1 class of dopamine-receptor-mediated stimulation of intracellular cAMP. CONCLUSION: Results may be of significance to understand the role of peripheral dopamine in human neuroimmune communication in terms of physiological homeostasis in health and disease.

Comparative study of antiplatelet drugs in vitro: distinct effects of cAMP-elevating drugs and GPIIb/IIIa antagonists on thrombin-induced platelet responses.

Among various categories of antiplatelet drugs, cAMP-elevating agents and GP IIb/IIIa antagonists have been reported to inhibit platelet aggregation stimulated by a wide variety of platelet agonists. To clarify the qualitative difference between these two agents, their effects on various platelet responses in washed platelets evoked by thrombin (0.05 U/mL) were compared in vitro. Two types of cAMP-elevating drugs, cilostazol (a phosphodiesterase III inhibitor) and prostaglandin E1 (an adenylate cyclase activator), both inhibited platelet aggregation, thromboxane A2 formation, and platelet factor 4 release in a concentration-dependent manner. In addition, both agents suppressed intracellular Ca++ elevation induced by thrombin. However, two classes of GP IIb/IIIa antagonists, abciximab (Fab fragment of antibody) and tirofiban (a synthetic compound), showed no inhibitory effects against thromboxane A2 formation and platelet factor 4 release, although these drugs inhibited platelet aggregation. Essentially the same results were obtained in platelet-rich plasma stimulated with high concentration (100 microM) of thrombin receptor activating peptide. In contrast to these different profiles on thromboxane A2 formation and release reaction, both cAMP-elevating agents and GP IIb/IIIa antagonists potently suppressed procoagulant activity in thrombin-stimulated platelets. These results suggest that the development of platelet procoagulant activity induced by thrombin is exclusively dependent on platelet aggregation or aggregation-dependent processes. These observations also indicate that cAMP-elevating agents possess wider inhibitory effects on platelet responses evoked by strong agonists than GP IIb/IIIa antagonists.

Ascorbic acid augments the adenylyl cyclase-cAMP system mediated POMC mRNA expression and beta-endorphin secretion from hypothalamic neurons in culture.

Besides acting as an important cofactor in the biosynthesis of catecholamine, ascorbic acid (AA) also modulates the activity of peptidylglycine-alpha-amidating monooxygenase for the post-translational modification of neuropeptides such as alpha-MSH and TRH. We report here a novel action of AA in modulating the secretion of immunoreactive beta-endorphin (ir-beta EP) and mRNA expression of proopiomelanocortin (POMC) following the activation of cAMP-dependent protein kinase A pathway in rat hypothalamic neurons. Primary cultures of hypothalamic neurons from neonatal rats as previously described were employed in the present studies. Six days after plating, cultures were replenished with serum-free media and incubated with vehicle or various doses of AA in the presence or absence of forskolin, 3-isobutyl-1-methylxanthine (IBMX), N6,2'-O-dibutyryladenosine 3'5'-(cyclic)monophosphate [(Bu)2cAMP]. Whereas the basal ir-beta EP release was 22.0 +/- 0.4 pg/well (mean +/- S.E.; n = 3), 10 microM of forskolin treatment increased ir-beta EP release approximately 4.2-fold. Co-incubation with AA enhanced forskolin induced ir-beta EP release and that this enhancing effect of AA was both time related and dose-dependent, with an ED50 of approximately 10 microM and an Emax of 100 microM. At the concentration of 10 microM, AA augmented ir-beta EP release approximately 6.1-fold that of cultures treated with forskolin alone. A similar potentiating effect of AA was also seen in cultures co-treated with IBMX or with (Bu)2cAMP. These enhancing effects of AA were similarly found in the abundance of total cAMP and of POMC mRNA of cultures which received identical treatments. However, it is important to point out that AA alone did not modulate ir-beta EP release or the abundance of POMC mRNA or total cAMP levels of the hypothalamic cultures when protein kinase A pathway was not activated. We thus conclude that AA augments cAMP-dependent protein kinase A pathway-induced production and release of beta EP from rat hypothalamic neurons in culture. Furthermore, this biological effect of AA is, at least in part, mediated through enhancing the responsiveness of the adenylyl cyclase-cAMP system.