Increase In Intracellular cAMP Levels Research Articles

The Effect of the cAMP Signaling Pathway on HTR8/SV-Neo Cell Line Proliferation, Invasion, and Migration After Treatment with Forskolin.

Pre-eclampsia (PE) is thought to be related to placental dysfunction, particularly poor extravillous trophoblast (EVT) invasion and migration abilities. However, the pathogenic mechanism is not fully understood. This article describes the impact of the cyclic adenosine monophosphate(cAMP) signaling pathway on EVT behavior, focusing on EVT proliferation, invasion, and migration. Here, we used the HTR8/SV-neo cell line to study human EVT function in vitro. HTR8/SV-neo cells were treated with different concentrations of forskolin (cAMP pathway-specific agonist) to alter intracellular cAMP levels, and dimethyl sulfoxide (DMSO) was used as the control. First, a cAMP assay was performed to measure the cAMP concentration in HTR8/SV-neo cells treated with different forskolin concentrations, and cell proliferation was assessed by constructing cell growth curves and assessing colony formation. Cell invasion and migration were observed by Transwell experiments, and intracellular epithelial-mesenchymal transition (EMT) marker expression was evaluated by quantitative real-time polymerase chain reaction (qPCR) and Western blotting (WB). According to our research, the intracellular cAMP levels in HTR8/SV-neo cells were increased in a dose-dependent manner, and HTR8/SV-neo cell proliferation, invasion and migration were significantly enhanced. The expression of EMT and angiogenesis markers was upregulated. Additionally, with the increase in intracellular cAMP levels, the phosphorylation of intracellular mitogen-activated protein kinase (MAPK) signaling pathway components was significantly increased. These results suggested that the cAMP signaling pathway promoted the phosphorylation of MAPK signaling components, thus enhancing EVT functions, including proliferation, invasion, and migration, and to a certain extent, providing a novel direction for the treatment of PE patients.

Pharmacological Evidence That Dictyostelium Differentiation-Inducing Factor 1 Promotes Glucose Uptake Partly via an Increase in Intracellular cAMP Content in Mouse 3T3-L1 Cells

Differentiation-inducing factor 1 (DIF-1) isolated from the cellular slime mold Dictyostelium discoideum can inhibit mammalian calmodulin-dependent cAMP/cGMP phosphodiesterase (PDE1) in vitro. DIF-1 also promotes glucose uptake, at least in part, via a mitochondria- and AMPK-dependent pathway in mouse 3T3-L1 fibroblast cells, but the mechanism underlying this effect has not been fully elucidated. In this study, we investigated the effects of DIF-1 on intracellular cAMP and cGMP levels, as well as the effects that DIF-1 and several compounds that increase cAMP and cGMP levels have on glucose uptake in confluent 3T3-L1 cells. DIF-1 at 20 μM (a concentration that promotes glucose uptake) increased the level of intracellular cAMP by about 20% but did not affect the level of intracellular cGMP. Neither the PDE1 inhibitor 8-methoxymethyl-3-isobutyl-1-methylxanthine at 10–200 μM nor the broad-range PDE inhibitor 3-isobutyl-1-methylxanthine at 40–400 μM had any marked effects on glucose uptake. The membrane-permeable cAMP analog 8-bromo-cAMP at 200–1000 μM significantly promoted glucose uptake (by 20–25%), whereas the membrane-permeable cGMP analog 8-bromo-cGMP at 3–100 μM did not affect glucose uptake. The adenylate cyclase activator forskolin at 1–10 μM promoted glucose uptake by 20–30%. Thus, DIF-1 may promote glucose uptake by 3T3-L1 cells, at least in part, via an increase in intracellular cAMP level.

OR09-05 GPBAR1- Understanding Its Function In The Secretory Endometrium

Abstract Disclosure: M. Saad-Naguib: None. P. Timmins: None. L. George: None. C. Samuels: None. N. Douglas: None. A.V. Babwah: None. Every successful pregnancy relies on implantation of an embryo into a receptive uterine endometrium. While implantation failure accounts for 50% of pregnancy failures, it remains the least understood aspect of pregnancy. In the mid-secretory (MS) phase of the menstrual cycle, under the influence of progesterone and cAMP, fibroblasts decidualize, a critical requirement for implantation. Decidualization involves the differentiation of stromal fibroblasts into secretory epithelioid cells that contribute to the formation of the decidua, the support of the pregnancy prior to placentation. A defective decidua can lead to implantation failure, but if implantation occurs, it could compromise placenta formation and function. Several studies have demonstrated the importance of G protein-coupled receptors (GPCRs) in mouse endometrial receptivity and decidualization. GPCRs are transmembrane signaling molecules that are ubiquitously expressed in the body and regulate almost every cellular and physiological process. They are major pharmaceutical targets, with 35% of all the drugs approved by the US FDA targeting GPCRs. To identify novel GPCR regulators of embryo implantation, we conducted bulk RNA sequencing of the human endometrium at the periovulatory (PO) and mid-secretory (MS) phase in natural menstrual cycles in healthy women of reproductive age and observed that genes encoding 11 GPCRs, not previously reported to regulate implantation, were differentially expressed between these two time points. Among these was the gene encoding the G protein-coupled bile acid receptor 1 (GPBAR1), and this was upregulated 4.6-fold in the MS vs PO phase. Single cell RNA sequence analysis showed that within the human endometrium, GPBAR1 mRNA expression is mostly and strongly localized to stromal fibroblasts. Upon activation by secondary bile acids, GPBAR1 couples to Gαs leading to an increase in intracellular cAMP levels. We hypothesize that in the MS endometrium, upon ligand binding, GPBAR1 produces cAMP and induces human endometrial stromal cell (HESC) decidualization. This hypothesis is being tested in vitro using primary HESCs. Interestingly, in the pregnant mouse, GPBAR1 mRNA is barely expressed at the implantation site on E7.5, and therefore unlikely plays a role in stromal cell decidualization in the mouse. Whether Gpbar1 is expressed in the pregnant mouse endometrium prior to implantation and plays a role in regulating endometrial receptivity is being investigated. Overall, our studies have uncovered a GPCR, that to our knowledge, has not been previously described in the human endometrium. Our data show that the expression of this GPCR is upregulated at the time of endometrial stromal cell decidualization, and therefore it might regulate decidualization and/or be required for decidual function in women. These possibilities are being examined. Presentation: Friday, June 16, 2023

93-LB: Discovery of ID110521156, a Small Molecule GLP-1 Receptor Agonist, for the Treatment of Type 2 Diabetes and Obesity

Glucagon-like peptide (GLP)-1 receptor agonists are widely used to treat type 2 diabetes and obesity by enhancing insulin release and suppressing appetite. Even semaglutide administrated orally has been developed, it still has limitation as a drug peptide, including high cost, less stability, and low bioavailability. This is the reason we aimed to discover a small molecule GLP-1 receptor agonist can be orally bioavailable. ID110521156 showed direct binding to GLP-1 receptor in a surface plasmon resonance assay. Treatment of ID110521156 induced a dose-dependent increase of intracellular cAMP levels in the human GLP-1 receptor expressed CHO cells with similar maximal efficacy with GLP-1 (7-36) amide. Partial agonism on the recruitment of β-arrestin was observed, whereas there are no effects on intracellular calcium levels, mitogen-activated protein kinase phosphorylation, and GLP-1 receptor internalization. Oral administration of ID110521156 demonstrated plasma glucose lowering and insulin secretion on intravenous glucose tolerance test using diabetic monkeys. In addition, we assured food intake reduction and body weight loss in a dose dependent manner by once daily dosing. Following oral administration, ID110521156 showed half-lives of 3.69-4.93 hours and bioavailability ranging 18-32% in normal cynomolgus monkeys. The no observed adverse effect level on the 28 days repeated dose toxicity study conducted in nonhuman primates was 100-fold margin with in vivo efficacy dose. ID110521156 demonstrated no liabilities when tested on a battery of safety pharmacology studies, including cardiovascular, respiratory, and neurobehavioral function assessment. No genotoxicity was observed on Ames, in vitro chromosomal aberration, and in vivo micronucleus test. From these nonclinical study results, ID110521156 might be considered as a potent candidate of small molecule selective GLP-1 receptor agonist to treat type 2 diabetes and obesity. Disclosure I. Je: None. K. An: None. H. Yoon: None. K. Kim: None. S. Choi: None. A. Im: None. D. Lee: None. W. Choi: None. E. Jang: None. J. Park: None. D. Kim: None. J. Lee: None. J. Park: None.

Functional Expression of IP, 5-HT4, D1, A2A, and VIP Receptors in Human Odontoblast Cell Line.

Odontoblasts are involved in sensory generation as sensory receptor cells and in dentin formation. We previously reported that an increase in intracellular cAMP levels by cannabinoid 1 receptor activation induces Ca2+ influx via transient receptor potential vanilloid subfamily member 1 channels in odontoblasts, indicating that intracellular cAMP/Ca2+ signal coupling is involved in dentinal pain generation and reactionary dentin formation. Here, intracellular cAMP dynamics in cultured human odontoblasts were investigated to understand the detailed expression patterns of the intracellular cAMP signaling pathway activated by the Gs protein-coupled receptor and to clarify its role in cellular functions. The presence of plasma membrane Gαs as well as prostaglandin I2 (IP), 5-hydroxytryptamine 5-HT4 (5-HT4), dopamine D1 (D1), adenosine A2A (A2A), and vasoactive intestinal polypeptide (VIP) receptor immunoreactivity was observed in human odontoblasts. In the presence of extracellular Ca2+, the application of agonists for the IP (beraprost), 5-HT4 (BIMU8), D1 (SKF83959), A2A (PSB0777), and VIP (VIP) receptors increased intracellular cAMP levels. This increase in cAMP levels was inhibited by the application of the adenylyl cyclase (AC) inhibitor SQ22536 and each receptor antagonist, dose-dependently. These results suggested that odontoblasts express Gs protein-coupled IP, 5-HT4, D1, A2A, and VIP receptors. In addition, activation of these receptors increased intracellular cAMP levels by activating AC in odontoblasts.

Capsaicin and Zinc Promote Glucose Uptake in C2C12 Skeletal Muscle Cells through a Common Calcium Signalling Pathway.

Capsaicin and zinc have recently been highlighted as potential treatments for glucose metabolism disorders; however, the effect of these two natural compounds on signalling pathways involved in glucose metabolism is still uncertain. In this study, we assessed the capsaicin- or zinc- induced activation of signalling molecules including calcium/calmodulin-dependent protein kinase 2 (CAMKK2), cAMP-response element-binding protein (CREB), and target of rapamycin kinase complex 1 (TORC1). Moreover, the expression status of genes associated with the control of glucose metabolism was measured in treated cells. The activation of cell signalling proteins was then evaluated in capsaicin- or zinc treated cells in the presence or absence of cell-permeant calcium chelator (BAPTA-AM) and the CAMKK inhibitor (STO-609). Finally, capsaicin- and zinc-induced glucose uptake was measured in the cells pre-treated with or without BAPTA-AM. Our results indicate that calcium flux induced by capsaicin or zinc led to activation of calcium signalling molecules and promoting glucose uptake in skeletal muscle cells. Pharmacological inhibition of CAMKK diminished activation of signalling molecules. Moreover, we observed an increase in intracellular cAMP levels in the cells after treatment with capsaicin and zinc. Our data show that capsaicin and zinc mediate glucose uptake in C2C12 skeletal muscle cells through the activation of calcium signalling.

Crosstalk between Drp1 phosphorylation sites during mitochondrial remodeling and their impact on metabolic adaptation

SummaryMitochondria constantly undergo fusion and fission events, referred as mitochondrial dynamics, which determine mitochondrial architecture and bioenergetics. Cultured cell studies demonstrate that mitochondrial dynamics are acutely regulated by phosphorylation of the mitochondrial fission orchestrator dynamin-related protein 1 (Drp1) at S579 or S600. However, the physiological impact and crosstalk of these phosphorylation sites is poorly understood. Here, we describe the functional interrelation between S579 and S600 phosphorylation sites in vivo and their role on mitochondrial remodeling. Mice carrying a homozygous Drp1 S600A knockin (Drp1 KI) mutation display larger mitochondria and enhanced lipid oxidation and respiratory capacities, granting improved glucose tolerance and thermogenic response upon high-fat feeding. Housing mice at thermoneutrality blunts these differences, suggesting a role for the brown adipose tissue in the protection of Drp1 KI mice against metabolic damage. Overall, we demonstrate crosstalk between Drp1 phosphorylation sites and provide evidence that their modulation could be used in the treatment and prevention of metabolic diseases.

Abstract 2951: Anti-GCGR in vivo efficacy evaluated in a novel humanized B-hGCGR mouse model

Abstract GCGR (glucagon receptor) is a G-protein-coupled seven-transmembrane protein, a typical representative of the class B GPCR family. Animals homozygous for a targeted mutation in this gene exhibit reduced blood glucose levels, increased plasma glucagon and amino acid levels associated with alpha-cell hyperplasia. Glucagon activates intracellular adenylate cyclase by specifically binding to GCGR on the surface of target cells in the liver. This results in an increase in intracellular cAMP levels, playing a role in promoting glycogenolysis and gluconeogenesis, and promoting blood glucose elevation. The crucial role in regulation of blood glucose levels and glucose homeostasis is posing GCGR as a potential drug target for type 2 diabetes and related diseases. Currently, much attention and many resources are directed towards research and development of antibody drugs targeting the glucagon receptor. Joining these efforts, Biocytogen has developed a GCGR humanized mouse model (B-hGCGR mouse). Human GCGR protein and mRNA were detected in liver tissue of homozygous B-hGCGR mice, but not in wild type mice. Physiological indexes have been analyzed. Results indicated that the random blood glucose, fasting blood glucose, and glucose tolerance were comparable to the wild type mice. In addition, we characterized the ability of glucagon to stimulate production of cAMP. We observed that glucagon elicits a dose specific increase of cAMP in membranes prepared from the humanized GCGR mice comparable to the cAMP levels observed in membranes prepared from wild type mice. In pharmacodynamic experiments using B-hGCGR mice we showed that an anti-GCGR antibody (Crotedumab) was able to effectively reduce random blood glucose and fasting blood glucose levels and improve the glucose tolerance, validating our humanized mouse model. This antibody also blocked the functional response to glucagon in B-hGCGR mice more efficiently than what can be observed in an approach targeting the wild type murine receptor using antagonists. These data demonstrate that B-hGCGR mice are a promising model for preclinical in vivo pharmacodynamic assessment of anti-GCGR antibodies. Citation Format: Rufeng Zhang, Chengzhang Shang, Yuting Hu, Veronika Chromikova, Qingcong Lin. Anti-GCGR in vivo efficacy evaluated in a novel humanized B-hGCGR mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2951.

Botrytis cinerea G Protein β Subunit Bcgb1 Controls Growth, Development and Virulence by Regulating cAMP Signaling and MAPK Signaling.

Botrytis cinerea is a necrotrophic phytopathogenic fungus that causes gray mold disease in many crops. To better understand the role of G protein signaling in the development and virulence of this fungus, the G protein β subunit gene Bcgb1 was knocked out in this study. The ΔBcgb1 mutants showed reduced mycelial growth rate, but increased aerial hyphae and mycelial biomass, lack of conidiation, failed to form sclerotia, increased resistance to cell wall and oxidative stresses, delayed formation of infection cushions, and decreased virulence. Deletion of Bcgb1 resulted in a significant reduction in the expression of several genes involved in cAMP signaling, and caused a notable increase in intracellular cAMP levels, suggesting that G protein β subunit Bcgb1 plays an important role in cAMP signaling. Furthermore, phosphorylation levels of MAP kinases (Bmp1 and Bmp3) were increased in the ΔBcgb1 mutants. Yeast two-hybrid assays showed that Bcgb1 interacts with MAPK (Bmp1 and Bmp3) cascade proteins (BcSte11, BcBck1, BcMkk1, and BcSte50), and the Bmp1-regulated gene Bcgas2 was up-regulated in the ΔBcgb1 mutant. These results indicated that Gβ protein Bcgb1 is involved in the MAPK signaling pathway in B. cinerea. In summary, our results revealed that Gβ protein Bcgb1 controls development and virulence through both the cAMP and MAPK (Bmp1 and Bmp3) signaling pathways in B. cinerea.

Opsin 3-Gαs Promotes Airway Smooth Muscle Relaxation Modulated by G Protein Receptor Kinase 2.

Recently, we characterized blue light-mediated relaxation (photorelaxation) of airway smooth muscle (ASM) and implicated the involvement of opsin 3 (OPN3), an atypical opsin. In the present study, we characterized the cellular signaling mechanisms of photorelaxation. We confirmed the functional role of OPN3 in blue light photorelaxation using trachea from OPN3 null mice (maximal relaxation 52 ± 13% compared with wild-type mice 90 ± 4.3%, P < 0.05). We then demonstrated colocalization of OPN3 and Gαs using co-IP and proximity ligation assays in primary human ASM cells, which was further supported by an increase in cAMP in mouse trachea treated with blue light compared with dark controls (23 ± 3.6 vs. 14 ± 2.6 pmol cAMP/ring, P < 0.05). Downstream PKA (protein kinase A) involvement was shown by inhibiting photorelaxation using Rp-cAMPS (P < 0.0001). Moreover, we observed converging mechanisms of desensitization by chronic β2-agonist exposure in mouse trachea and correlated this finding with colocalization of OPN3 and GRK2 (G protein receptor kinase) in primary human ASM cells. Finally, an overexpression model of OPN1LW (a red light photoreceptor in the same opsin family) in human ASM cells showed an increase in intracellular cAMP levels following red light exposure compared with nontransfected cells (48 ± 13 vs. 13 ± 2.1 pmol cAMP/mg protein, P < 0.01), suggesting a conserved photorelaxation mechanism for wavelengths of light that are more tissue penetrant. Together, these results demonstrate that blue light photorelaxation in ASM is mediated by the OPN3 receptor interacting with Gαs, which increases cAMP levels, activating PKA and modulated by GRK2.

A non-catalytic function of PI3Kγ drives smooth muscle cell proliferation after arterial damage.

Arterial remodeling in hypertension and intimal hyperplasia involves inflammation and disrupted flow, both of which contribute to smooth muscle cell dedifferentiation and proliferation. In this context, our previous results identified phosphoinositide 3-kinase γ (PI3Kγ) as an essential factor in inflammatory processes of the arterial wall. Here, we identify for the first time a kinase-independent role of nonhematopoietic PI3Kγ in the vascular wall during intimal hyperplasia using PI3Kγ-deleted mice and mice expressing a kinase-dead version of the enzyme. Moreover, we found that the absence of PI3Kγ in vascular smooth muscle cells (VSMCs) leads to modulation of cell proliferation, associated with an increase in intracellular cAMP levels. Real-time analysis of cAMP dynamics revealed that PI3Kγ modulates the degradation of cAMP in primary VSMCs independently of its kinase activity through regulation of the enzyme phosphodiesterase 4. Importantly, the use of an N-terminal competing peptide of PI3Kγ blocked primary VSMC proliferation. These data provide evidence for a kinase-independent role of PI3Kγ in arterial remodeling and reveal novel strategies targeting the docking function of PI3Kγ for the treatment of cardiovascular diseases.

A novel secreted-cAMP pathway inhibits pulmonary hypertension via a feed-forward mechanism.

Cyclic adenosine monophosphate (cAMP) is the predominant intracellular second messenger that transduces signals from Gs-coupled receptors. Intriguingly, there is evidence from various cell types that an extracellular cAMP pathway is active in the extracellular space. Herein, we investigated the role of extracellular cAMP in the lung and examined whether it may act on pulmonary vascular cell proliferation and pulmonary vasculature remodelling in the pathogenesis of pulmonary hypertension (PH). The expression of cyclic AMP-metabolizing enzymes was increased in lungs from patients with PH as well as in rats treated with monocrotaline and mice exposed to Sugen/hypoxia. We report that inhibition of the endogenous extracellular cAMP pathway exacerbated Sugen/hypoxia-induced lung remodelling. We found that application of extracellular cAMP induced an increase in intracellular cAMP levels and inhibited proliferation and migration of pulmonary vascular cells in vitro. Extracellular cAMP infusion in two in vivo PH models prevented and reversed pulmonary and cardiac remodelling associated with PH. Using protein expression analysis along with luciferase assays, we found that extracellular cAMP acts via the A2R/PKA/CREB/p53/Cyclin D1 pathway. Taken together, our data reveal the presence of an extracellular cAMP pathway in pulmonary arteries that attempts to protect the lung during PH, and suggest targeting of the extracellular cAMP signalling pathway to limit pulmonary vascular remodelling and PH.

NaCl triggers the CRP-dependent increase of cAMP in Mycobacterium tuberculosis

The second messenger 3′,5′-cyclic adenosine monophosphate (3′,5′-cAMP) has been shown to be involved in the regulation of many biological processes ranging from carbon catabolite repression in bacteria to cell signalling in eukaryotes. In mycobacteria, the role of cAMP and the mechanisms utilized by the bacterium to adapt to and resist immune and pharmacological sterilization remain poorly understood. Among the stresses encountered by bacteria, ionic and non-ionic osmotic stresses are among the best studied. However, in mycobacteria, the link between ionic osmotic stress, particularly sodium chloride, and cAMP has been relatively unexplored. Using a targeted metabolic analysis combined with stable isotope tracing, we show that the pathogenic Mycobacterium tuberculosis but not the opportunistic pathogen Mycobacterium marinum nor the non-pathogenic Mycobacterium smegmatis responds to NaCl stress via an increase in intracellular cAMP levels. We further showed that this increase in cAMP is dependent on the cAMP receptor protein and in part on the threonine/serine kinase PnkD, which has previously been associated with the NaCl stress response in mycobacteria.

Transcriptomic analysis of Spodoptera frugiperda Sf9 cells resistant to Bacillus thuringiensis Cry1Ca toxin reveals that extracellular Ca2+, Mg2+ and production of cAMP are involved in toxicity.

ABSTRACTBacillus thuringiensis (Bt) produces pore forming toxins that have been used for pest control in agriculture for many years. However, their molecular and cellular mode of action is still unclear. While a first model – referred to as the pore forming model – is the most widely accepted scenario, a second model proposed that toxins could trigger an Mg2+-dependent intracellular signalling pathway leading to cell death. Although Cry1Ca has been shown to form ionic pores in the plasma membrane leading to cell swelling and death, we investigated the existence of other cellular or molecular events involved in Cry1Ca toxicity. The Sf9 insect cell line, derived from Spodoptera frugiperda, is highly and specifically sensitive to Cry1Ca. Through a selection program we developed various levels of laboratory-evolved Cry1Ca-resistant Sf9 cell lines. Using a specific S. frugiperda microarray we performed a comparative transcriptomic analysis between sensitive and resistant cells and revealed genes differentially expressed in resistant cells and related to cation-dependent signalling pathways. Ion chelators protected sensitive cells from Cry1Ca toxicity suggesting the necessity of both Ca2+ and/or Mg2+ for toxin action. Selected cells were highly resistant to Cry1Ca while toxin binding onto their plasma membrane was not affected. This suggested a resistance mechanism different from the classical ‘loss of toxin binding’. We observed a correlation between Cry1Ca cytotoxicity and the increase of intracellular cAMP levels. Indeed, Sf9 sensitive cells produced high levels of cAMP upon toxin stimulation, while Sf9 resistant cells were unable to increase their intracellular cAMP. Together, these results provide new information about the mechanism of Cry1Ca toxicity and clues to potential resistance factors yet to discover.

Protein kinase A inhibition induces EPAC-dependent acrosomal exocytosis in human sperm.

To interact with the egg, the spermatozoon must undergo several biochemical and motility modifications in the female reproductive tract, collectively called capacitation. Only capacitated sperm can undergo acrosomal exocytosis, near or on the egg, a process that allows the sperm to penetrate and fertilize the egg. In the present study, we investigated the involvement of cyclic adenosine monophosphate (cAMP)-dependent processes on acrosomal exocytosis. Inhibition of protein kinase A (PKA) at the end of capacitation induced acrosomal exocytosis. This process is cAMP-dependent; however, the addition of relatively high concentration of the membrane-permeable 8-bromo-cAMP (8Br-cAMP, 0.1 mmol l−1) analog induced significant inhibition of the acrosomal exocytosis. The induction of acrosomal exocytosis by PKA inhibition was significantly inhibited by an exchange protein directly activated by cAMP (EPAC) ESI09 inhibitor. The EPAC selective substrate activated AE at relatively low concentrations (0.02–0.1 μmol l−1), whereas higher concentrations (>5 μmol l−1) were inhibitory to the AE induced by PKA inhibition. Inhibition of PKA revealed about 50% increase in intracellular cAMP levels, conditions under which EPAC can be activated to induce the AE. Induction of AE by activating the actin severing-protein, gelsolin, which causes F-actin dispersion, was inhibited by the EPAC inhibitor. The AE induced by PKA inhibition was mediated by phospholipase C activity but not by the Ca2+-channel, CatSper. Thus, inhibition of PKA at the end of the capacitation process induced EPAC/phospholipase C-dependent acrosomal exocytosis. EPAC mediates F-actin depolymerization and/or activation of effectors downstream to F-actin breakdown that lead to acrosomal exocytosis.

Bicarbonate Inhibits Bacterial Growth and Biofilm Formation of Prevalent Cystic Fibrosis Pathogens.

We investigated the effects of bicarbonate on the growth of several different bacteria as well as its effects on biofilm formation and intracellular cAMP concentration in Pseudomonas aeruginosa. Biofilm formation was examined in 96-well plates, with or without bicarbonate. The cAMP production of bacteria was measured by a commercial assay kit. We found that NaHCO3 (100 mmol l-1) significantly inhibited, whereas NaCl (100 mmol l-1) did not influence the growth of planktonic bacteria. MIC and MBC measurements indicated that the effect of is bacteriostatic rather than bactericidal. Moreover, NaHCO3 prevented biofilm formation as a function of concentration. Bicarbonate and alkalinization of external pH induced a significant increase in intracellular cAMP levels. In conclusion, impedes the planktonic growth of different bacteria and impedes biofilm formation by P. aeruginosa that is associated with increased intracellular cAMP production. These findings suggest that aerosol inhalation therapy with solutions may help improve respiratory hygiene in patients with cystic fibrosis and possibly other chronically infected lung diseases.

Critical role of phosphodiesterase 2A in mouse congenital heart defects.

Phosphodiesterase 2 A (Pde2A), a cAMP-hydrolysing enzyme, is essential for mouse development; however, the cause of Pde2A knockout embryonic lethality is unknown. To understand whether Pde2A plays a role in cardiac development, hearts of Pde2A deficient embryos were analysed at different stage of development. At the stage of four chambers, Pde2A deficient hearts were enlarged compared to the hearts of Pde2A heterozygous and wild-type. Pde2A knockout embryos revealed cardiac defects such as absence of atrial trabeculation, interventricular septum (IVS) defects, hypertrabeculation and thinning of the myocardial wall and in rare cases they had overriding aorta and valves defects. E14.5 Pde2A knockouts showed reduced cardiomyocyte proliferation and increased apoptosis in the IVS and increased proliferation in the ventricular trabeculae. Analyses of E9.5 Pde2A knockout embryos revealed defects in cardiac progenitor and neural crest markers, increase of Islet1 positive and AP2 positive apoptotic cells. The expression of early cTnI and late Mef2c cardiomyocyte differentiation markers was strongly reduced in Pde2A knockout hearts. The master transcription factors of cardiac development, Tbx, were down-regulated in E14.5 Pde2A knockout hearts. Absence of Pde2A caused an increase of intracellular cAMP level, followed by an up-regulation of the inducible cAMP early repressor, Icer in fetal hearts. In vitro experiments on wild-type fetal cardiomyocytes showed that Tbx gene expression is down-regulated by cAMP inducers. Furthermore, Pde2A inhibition in vivo recapitulated the heart defects observed in Pde2A knockout embryos, affecting cardiac progenitor cells. Interestingly, the expression of Pde2A itself was dramatically affected by Pde2A inhibition, suggesting a potential autoregulatory loop. We demonstrated for the first time a direct relationship between Pde2A impairment and the onset of mouse congenital heart defects, highlighting a novel role for cAMP in cardiac development regulation.

Abstract 178: Peptide nanofibers: targeted therapies for glioblastoma multiforme

Abstract Glioblastoma multiforme (GBM) is a malignant brain tumor with poor prognosis due to tumor heterogeneity, poor drug blood-brain barrier (BBB) permeability and targeting. GBM biopsies indicated the overexpression of G-protein coupled receptors (GPCRs) that when activated by neuropeptide agonists result in an antiproliferative effect. However, translation of these neuropeptides into novel therapies for GBM is frustrated by their short half-life (&amp;lt;5 minutes) and inability to cross the BBB. Here, we present a novel strategy based on the lipidization of peptide agonists which results in peptide amphiphiles able to self-assemble into nanofibers that can entrap brain impermeable drugs, possess an enhanced stability to enzymatic degradation, permeate across an all human in vitro BBB model and are able to target GBM cells resulting in a significant antiproliferative effect. Peptide amphiphiles were synthesized using solid-phase peptide synthesis and characterised using pyrene, thioflavin T, circular dichroism and transmission electron microscopy experiments. Nanofibers were loaded with brain impermeable cytotoxic drugs (e.g. paclitaxel) and their stability was studied in biological media (plasma, brain, liver and GBM cell lysates). BBB permeation was studied in an all human in vitro Transwell model. The antiproliferative effect of the nanofibers was evaluated on U-87 MG cells. Targeting of the nanofibers to the GPCR was evaluated using single-molecule force spectroscopy (SMFS). Peptide amphiphiles self-assemble into stable nanofibers at concentrations above 189 μM and are able to solubilize high amounts of paclitaxel (&amp;gt;1.8 mg) ensuring that the resulting nanomedicine can be clinically translatable. Both unloaded- and paclitaxel loaded-nanofibers showed superior stability compared to the parent neuropeptide in the presence of plasma, brain, liver and cell homogenates (&amp;gt;6-fold). Nanofibers elicited a strong antiproliferative effect (IC50: 5.06 μM) resulting in a cell cycle arrest at G2/M in a GPCR positive GBM cell line (U-87 MG). Loading paclitaxel (1nM) within the nanofibers resulted in a synergistic effect evidenced by a decrease in cell survival by 32%. Nanofibers counteracted the forskolin-induced increase of intracellular cAMP levels indicating that the GPCR is linked to the GαI protein known to mediate the antiproliferative effect on GBM. Confocal studies confirmed the internalization of the peptide while SMFS studies supported specific binding of the nanofibers to GPCR on U-87MG cell surface with equivalent binding probability to peptide agonists and increased residence time. Texas Red labelled nanofibers permeate across an in vitro BBB model enabling the permeation of paclitaxel (Papp: 8.45 x10-6 cm/s). Thus, described peptide nanofibers are a novel targeted nanomedicine for GBM therapy able to be clinically translated. In vivo proof of concept studies and pharmacokinetics are under way. Citation Format: Diana M. Leite, Rong Zhu, Eugen Barbu, Peter Hinterdorfer, Geoffrey J. Pilkington, Aikaterini Lalatsa. Peptide nanofibers: targeted therapies for glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 178. doi:10.1158/1538-7445.AM2017-178

TUDCA: An Agonist of the Bile Acid Receptor GPBAR1/TGR5 With Anti-Inflammatory Effects in Microglial Cells.

Bile acids are steroid acids found in the bile of mammals. The bile acid conjugate tauroursodeoxycholic acid (TUDCA) is neuroprotective in different animal models of stroke and neurological diseases. We have previously shown that TUDCA has anti-inflammatory effects on glial cell cultures and in a mouse model of acute neuroinflammation. We show now that microglial cells (central nervous system resident macrophages) express the G protein-coupled bile acid receptor 1/Takeda G protein-coupled receptor 5 (GPBAR1/TGR5) in vivo and in vitro. TUDCA binding to GPBAR1/TGR5 caused an increase in intracellular cAMP levels in microglia that induced anti-inflammatory markers, while reducing pro-inflammatory ones. This anti-inflammatory effect of TUDCA was inhibited by small interference RNA for GPBAR1/TGR5 receptor, as well as by treatment with a protein kinase A (PKA) inhibitor. In the mouse model of acute neuroinflammation, treating the animals with TUDCA was clearly anti-inflammatory. TUDCA biased the microglial phenotype in vivo and in vitro toward the anti-inflammatory. The bile acid receptor GPBAR1/TGR5 could be a new therapeutic target for pathologies coursing with neuroinflammation and microglia activation, such as traumatic brain injuries, stroke, or neurodegenerative diseases. TUDCA and other GPBAR1/TGR5 agonists need to be further investigated, to determine their potential in attenuating the neuropathologies associated with microglia activation. J. Cell. Physiol. 232: 2231-2245, 2017. © 2016 Wiley Periodicals, Inc.

Identification and functional characterisation of 5-HT4 receptor in sea cucumber Apostichopus japonicus (Selenka)

Serotonin (5-HT) is an important neurotransmitter and neuromodulator that controls a variety of sensory and motor functions through 5-HT receptors (5-HTRs). The 5-HT4R subfamily is linked to Gs proteins, which activate adenylyl cyclases (ACs), and is involved in many responses in peripheral organs. In this study, the 5-HT4R from Apostichopus japonicus (Aj5-HT4R) was identified and characterised. The cloned full-length Aj5-HT4R cDNA is 1,544 bp long and contains an open reading frame 1,011 bp in length encoding 336 amino acid proteins. Bioinformatics analysis of the Aj5-HT4R protein indicated this receptor was a member of class A G protein coupled receptor (GPCR) family. Further experiments using Aj5-HT4R-transfected HEK293 cells demonstrated that treatment with 5-HT triggered a significant increase in intracellular cAMP level in a dose-dependent manner and induced a rapid internalisation of Aj5-HT4R fused with enhanced green fluorescent protein (Aj5-HT4R-EGFP) from the cell surface into the cytoplasm. In addition, the transcriptional profiles of Aj5-HT4R in aestivating A. japonicas and phosphofructokinase (AjPFK) in 5-HT administrated A. japonicus have been analysed by real-time PCR assays. Results have led to a basic understanding of Aj5-HT4R in A. japonicus, and provide a foundation for further exploration of the cell signaling and regulatory functions of this receptor.