Intracellular Cyclic AMP Levels Research Articles

The impact of glucagon to support postabsorptive glucose flux and glycemia in healthy rats and its attenuation in male Zucker diabetic fatty rats.

Hyperglucagonemia is a hallmark of type 2 diabetes (T2DM), yet the role of elevated plasma glucagon (P-GCG) to promote excessive postabsorptive glucose production and contribute to hyperglycemia in patients with this disease remains debatable. We investigated the acute action of P-GCG to safeguard/support postabsorptive endogenous glucose production (EGP) and euglycemia in healthy Zucker control lean (ZCL) rats. Using male Zucker diabetic fatty (ZDF) rats that exhibit the typical metabolic disorders of human T2DM, such as excessive EGP, hyperglycemia, hyperinsulinemia, and hyperglucagonemia, we examined the ability of hyperglucagonemia to promote greater rates of postabsorptive EGP and hyperglycemia. Euglycemic or hyperglycemic basal insulin (INS-BC) and glucagon (GCG-BC) clamps were performed in the absence or during an acute setting of glucagon deficiency (GCG-DF, ∼10% of basal), either alone or in combination with insulin deficiency (INS-DF, ∼10% of basal). Glucose appearance, disappearance, and cycling rates were measured using [2-3H] and [3-3H]-glucose. In ZCL rats, GCG-DF reduced the levels of hepatic cyclic AMP, EGP, and plasma glucose (PG) by 50%, 32%, and 50%, respectively. EGP fell in the presence GCG-DF and INS-BC, but under GCG-DF and INS-DF, EGP and PG increased two- and threefold, respectively. GCG-DF revealed the hyperglucagonemia present in ZDF rats lacked the ability to regulate hepatic intracellular cyclic AMP levels and glucose flux, since EGP and PG levels fell by only 10%. We conclude that the liver in T2DM suffers from resistance to all three major regulatory factors, glucagon, insulin, and glucose, thus leading to a loss of metabolic flexibility.NEW & NOTEWORTHY In postabsorptive state, basal plasma insulin (P-INS) and plasma glucose (PG) act dominantly to increase hepatic glucose cycling and reduce endogenous glucose production (EGP) and PG in healthy rats, which is only counteracted by the acute action of basal plasma glucagon (P-GCG) to support EGP and euglycemia. Hyperglucagonemia, a hallmark of type 2 diabetes (T2DM) present in Zucker diabetic fatty (ZDF) rats, is not the primary mediator of hyperglycemia and high EGP as commonly thought; instead, the liver is resistant to glucagon as well as insulin and glucose.

Salt-inducible kinases regulate androgen synthesis in theca cells by enhancing CREB signaling

Ovulation is the pinnacle of folliculogenesis, a process that requires an interplay between the oocyte, the granulosa cells, and the theca cells (TCs). TCs are the only source of ovarian androgens, which play a vital role in female fertility. However, abnormally elevated androgen levels reduce fertility. Therefore, uncovering novel mechanisms regulating androgen synthesis in TCs is of great significance. We have shown that salt-inducible kinases (SIKs) regulate granulosa cell steroidogenesis. Here, we investigated whether SIKs regulate androgen production in TCs. SIK2 and SIK3 were detected in the TCs of mouse ovaries and isolated TCs. Next, TCs in culture were treated with luteinizing hormone (LH) in the presence or absence of a highly specific SIK inhibitor. SIK inhibition enhanced the stimulatory effect of LH on steroidogenic gene expression and androgen production in a concentration-dependent manner. SIK inhibition alone stimulated the expression of steroidogenic genes and increased androgen production. Activation of adenylyl cyclase with forskolin or emulation of increased intracellular cyclic AMP levels stimulated steroidogenesis, an effect that was enhanced by the inhibition of SIK activity. The stimulatory effect of downstream targets of cyclic AMP was also significantly augmented by SIK inhibition, suggesting that SIKs control targets downstream cyclic AMP. Finally, it is shown that SIK2 knockout mice have higher circulating testosterone than controls. This evidence shows that TCs express SIKs and reveal novel roles for SIKs in the regulation of TC function and androgen production. This information could contribute to uncovering therapeutic targets to treat hyperandrogenic diseases.

Impact of the cAMP efflux and extracellular cAMP-adenosine pathway on airway smooth muscle relaxation induced by formoterol and phosphodiesterase inhibitors

β2-adrenoceptors agonists and phosphodiesterase (PDE) inhibitors are effective bronchodilators, due to their ability to increase intracellular cyclic AMP (cAMP) levels and induce airway smooth muscle (ASM) relaxation. We have shown that increment of intracellular cAMP induced by β2-adrenoceptors agonist fenoterol is followed by efflux of cAMP, which is converted by ecto-PDE and ecto-5′-nucleotidases (ecto-5′NT) to adenosine, leading to ASM contraction. Here we evaluate whether other classical bronchodilators used to treat asthma and chronic obstructive pulmonary disease (COPD) could induce cAMP efflux and, as consequence, influence the ASM contractility. Our results showed that β2-adrenoceptor agonists formoterol and PDE inhibitors IBMX, aminophylline and roflumilast induced cAMP efflux and a concentration-dependent relaxation of rat trachea precontracted with carbachol. Pretreatment of tracheas with MK-571 (MRP transporter inhibitor), AMP-CP (ecto-5′NT inhibitor) or CGS-15943 (nonselective adenosine receptor antagonist) potentiated the relaxation induced by β2-adrenoceptor agonists but did not change the relaxation induced by PDE inhibitors. These data showed that all bronchodilators tested were able to induce cAMP efflux. However, only β2-adrenoceptor-induced relaxation of tracheal smooth muscle was affected by cAMP efflux and extracellular cAMP-adenosine pathway.

Association of a single amino acid replacement with dorsal pigmentation in a lizard from the Qinghai-Tibetan Plateau

Reptiles can evolve adaptive colors in different environments, but relatively little is known about the genetic mechanisms. Here, we identified the MC1R gene and its association with intraspecific color variation in the lizard Phrynocephalus erythrurus. Analysis of the MC1R sequence in 143 individuals from dark South Qiangtang Plateau (SQP) and light North Qiangtang plateau (NQP) populations, revealed two amino acid sites that showed significant differences in frequency between two areas. One SNP, corresponding to Glu183Lys residue, was found to be a highly significant outlier and differentially fixed for SQP and NQP populations. This residue is located in an extracellular area in the second small extracellular loop within the secondary structure of MC1R, which represents an “attachment pocket” part of the 3D structure. Cytological expression of MC1R alleles with the Glu183Lys replacement showed a 39 % increase in intracellular agonist-induced cyclic AMP levels and a 23.18 % greater cell surface expression of MC1R protein in the SQP relative to the NQP allele. Further in silico 3D modeling and in vitro binding experiments indicated a higher MC1R-α-MSH binding for the SQP allele, and elevated melanin synthesis. We provide an overview of how a single amino acid replacement leads to fundamental changes in MC1R function, and hence shapes variation in dorsal pigmentation in lizards from different environments.

Relaxin-like Gonad-Stimulating Peptides in Asteroidea.

Starfish relaxin-like gonad-stimulating peptide (RGP) is the first identified peptide hormone with gonadotropin-like activity in invertebrates. RGP is a heterodimeric peptide, comprising A and B chains with disulfide cross-linkages. Although RGP had been named a gonad-stimulating substance (GSS), the purified peptide is a member of relaxin-type peptide family. Thus, GSS was renamed as RGP. The cDNA of RGP encodes not only the A and B chains, but also signal and C-peptides. After the rgp gene is translated as a precursor, mature RGP is produced by eliminating the signal and C-peptides. Hitherto, twenty-four RGP orthologs have been identified or predicted from starfish in the orders Valvatida, Forcipulatida, Paxillosida, Spinulosida, and Velatida. The molecular evolution of the RGP family is in good accordance with the phylogenetic taxonomy in Asteroidea. Recently, another relaxin-like peptide with gonadotropin-like activity, RLP2, was found in starfish. RGP is mainly present in the radial nerve cords and circumoral nerve rings, but also in the arm tips, the gonoducts, and the coelomocytes. RGP acts on ovarian follicle cells and testicular interstitial cells to induce the production of 1-methyladenine (1-MeAde), a starfish maturation-inducing hormone. RGP-induced 1-MeAde production is accompanied by an increase in intracellular cyclic AMP levels. This suggests that the receptor for RGP (RGPR) is a G protein-coupled receptor (GPCR). Two types of GPCRs, RGPR1 and RGPR2, have been postulated as candidates. Furthermore, 1-MeAde produced by RGP not only induces oocyte maturation, but also induces gamete shedding, possibly by stimulating the secretion of acetylcholine in the ovaries and testes. Thus, RGP plays an important role in starfish reproduction, but its secretion mechanism is still unknown. It has also been revealed that RGP is found in the peripheral adhesive papillae of the brachiolaria arms. However, gonads are not developed in the larvae before metamorphosis. It may be possible to discover new physiological functions of RGP other than gonadotropin-like activity.

Defibrotide Inhibits Antiphospholipid Antibody-Mediated Neutrophil Extracellular Trap Formation and Venous Thrombosis.

Defibrotide is a heterogenous mixture of polyanionic oligonucleotides currently approved for treatment of transplant-associated venoocclusive disease. While defibrotide has a known role in limiting endothelial cell activation, some studies have also demonstrated anti-leukocyte properties. In a recent study, we found that neutrophil extracellular traps (NETs) play a role in the thrombotic complications of antiphospholipid syndrome (APS). In the present study, we investigated the hypothesis that defibrotide might act to mitigate APS-relevant NET formation in vitro and in mouse models. We used in vitro assays and a mouse model to determine the mechanisms by which defibrotide inhibits NET formation and venous thrombosis in APS. At doses ranging from 1 to 10 μg/ml, defibrotide significantly suppressed NET formation from control neutrophils stimulated with IgG isolated from patients with APS. Defibrotide increased levels of intracellular cyclic AMP in neutrophils, and its suppressive effects on NET formation were mitigated by blocking adenosine A2A receptor or by inhibiting the cyclic AMP-dependent kinase protein kinase A. Defibrotide at doses ranging from 15 to 150 mg/kg/day inhibited NET formation and venous thrombosis in a model of antiphospholipid antibody-accelerated thrombosis-an effect that was reduced in adenosine A2A receptor-knockout mice. This study is the first to demonstrate mechanisms by which defibrotide counteracts neutrophil-mediated thrombotic inflammation inherent to APS.

Apremilast in the Treatment of Plaque Psoriasis: Differential Use in Psoriasis.

Small molecule medications like apremilast are emerging as promising options for patients with psoriasis and other inflammatory conditions. Apremilast was approved by the Food and Drug Administration in 2014 for the management of both psoriasis and psoriatic arthritis. Apremilast inhibits phosphodiesterase-4, which increases the intracellular levels of cyclic AMP, thereby reducing inflammatory cytokine production. This review aims to discuss the published evidence and evaluate the differential use of apremilast in plaque psoriasis of the body and scalp, nail psoriasis, and palmoplantar psoriasis. In clinical trials, apremilast effectively reduced the severity of different dermatological manifestations of psoriasis and improved patients’ quality of life. It has an acceptable safety profile and is generally well-tolerated. Oral medications like apremilast offer an alternative route of administration which can be more convenient and appropriate for some patients. Additionally, pharmacoeconomic analyses of available anti-psoriatic systemic agents favor apremilast as a cost-effective therapeutic option.

Immunosuppression and Increased Risk of Bacterial Infections in Cirrhosis

Immunosuppression and Increased Risk of Bacterial Infections in Cirrhosis

P–098 Use of Dimethylxanthine Theophylline in surgical retrieved sperms that do not recover motility after thawing

Abstract Study question Can the use of Theophylline recover motility of frozen surgically retrieved sperms in case of absence of motility after thawing? Summary answer Theophylline allows to recover motility of thawed surgically retrieved sperms. The utilization of sperms with or without pharmacological activation gives comparable clinical outcomes. What is known already Testicular sperm motility is usually poor. A method is needed to detect viable sperm for ICSI when motility is totally absent after freezing/thawing. Hypo-osmotic swelling test, mechanical touch technique, laser-assisted immotile sperm selection, birefringence-polarization microscopy and exposure to pharmacological stimulation are techniques used for this purpose. Among pharmacological agents Dimethylxanthine Theophylline is a phosphodiesterase inibitor that improves sperm motility by promoting an increase in intracellular cyclic AMP levels. Few studies report that it is efficient for recovery of sperm motility in cases of thawed testicular and retrograde ejaculation samples improving reproductive outcomes. Study design, size, duration Retrospective analysis of sixty frozen surgical sperm cycles (45 patients) utilized from February 2018 to November 2020. After thawing, samples were divided in two Groups according to motility recovery. Group A: presence of motility, Group B: absence of motility. Group B was treated with Theophilline and motility was re-assessed after incubation. Activated sperms were utilized for ICSI when available. Sperm motility recovery, fertilization, pregnancy rate/transfer, implantation and miscarriage rate were evaluated in both Groups. Participants/materials, setting, methods Surgical specimens were treated and concentrated in SpermRinse™ Medium (Vitrolife) and then cryopreservated in nitrogen vapor in TEST Yolk Buffer (Irvine Scientific). After thawing, only samples with no motility recovery were treated with a brief incubation in Theophylline (GM501 SpermMobil, Gynemed) and washed in Polyvinylpyrrolidone (ICSITM Vitrolife) before injection. ICSI was performed in all cases approximately 4–5 hours after sperm thawing. After fertilization check, transfer was scheduled in day 2. Main results and the role of chance Women’s age Group A (34,39±2,29 M±SD) and group B (35,87±4,34 M±SD) and men’s age Group A (37,31±5,12 M±SD) and group B (40,89±8.15 M±SD) were not significantly different (P= .328 and P=.218) respectively. Group A: 13/60 cycles (21.7%) (9 patients). Pre freezing and post thawing total motility percentage were 34.0±19.0 (M±SD) and 13.5±15.6 (M±SD) respectively (39.8% recovery). Group B: 47/60 cycles (78.3%) (36 patients). Pre freezing total motility percentage was 5.3±8.5 (M±SD) and no motility was recovered post thawing (0%). After treatment with Theophylline total motility was 1.8±1.8 (M±SD) (33.5% recovery). Motile sperms were utilized in all cases except from two in the Group B. Number of injected oocytes was 2.8±1.1 (M±SD) in Group A and 4.3±3.1 (M±SD) in Group B (P=.004) respectively. Fertilisation rate (63.1% and 45.4%, P=.066), Number of embryos transferred (1.8±0.7 M±SD and 1.6±0.7 M±SD, P=.271), Pregnancy rate/Transfer (54.5% and 37.1%, P=.502), Implantation rate (30.0% and 27.8%, P=.919) and Miscarriage rate (33.3% and 30.7%, P=.675) were not statistically significant between Group A and B respectively. In the two cases of group B injected with immotile sperm, fertilization rate was 0% (0/3) and 50% (2/4). Limitations, reasons for caution A larger study is needed to investigate the recovery of sperms motility (and/or their activation) and clinical outcomes, in particular referring to the origin of sampling (epididymal aspirate and testicular tissue) and type of azoospermia (obstructive and non-obstructive). Wider implications of the findings: Theophylline is an effective tool for sperm motility recovery after thawing allowing to inject viable sperm and facilitating laboratory handling. Trial registration number Not applicable

Abiraterone Acetate Induces CREB1 Phosphorylation and Enhances the Function of the CBP-p300 Complex, Leading to Resistance in Prostate Cancer Cells.

Abiraterone acetate (AA), an inhibitor of cytochrome P450 17alpha-hydroxylase/17, 20 lyase, is an FDA-approved drug for advanced prostate cancer. However, not all patients respond to AA, and AA resistance ultimately develops in patients who initially respond. We aimed to identify AA resistance mechanisms in prostate cancer cells. We established several AA-resistant cell lines and performed a comprehensive study on mechanisms involved in AA resistance development. RNA sequencing and phospho-kinase array screenings were performed to discover that the cAMP-response element CRE binding protein 1 (CREB1) was a critical molecule in AA resistance development. The drug-resistant cell lines are phenotypically stable without drug selection, and exhibit permanent global gene expression changes. The phosphorylated CREB1 (pCREB1) is increased in AA-resistant cell lines and is critical in controlling global gene expression. Upregulation of pCREB1 desensitized prostate cancer cells to AA, while blocking CREB1 phosphorylation resensitized AA-resistant cells to AA. AA treatment increases intracellular cyclic AMP (cAMP) levels, induces kinases activity, and leads to the phosphorylation of CREB1, which may subsequently augment the essential role of the CBP/p300 complex in AA-resistant cells because AA-resistant cells exhibit a relatively higher sensitivity to CBP/p300 inhibitors. Further pharmacokinetics studies demonstrated that AA significantly synergizes with CBP/p300 inhibitors in limiting the growth of prostate cancer cells. Our studies suggest that AA treatment upregulates pCREB1, which enhances CBP/p300 activity, leading to global gene expression alterations, subsequently resulting in drug resistance development. Combining AA with therapies targeting resistance mechanisms may provide a more effective treatment strategy.

Hydrogen sulfide induces Ca2+ release from the endoplasmic reticulum and suppresses ATP-induced Ca2+ signaling in rat spinal cord astrocytes

Hydrogen sulfide (H2S) has a variety of physiological functions. H2S reportedly increases intracellular Ca2+ concentration ([Ca2+]i) in astrocytes. However, the precise mechanism and functional role of this increase are not known. Here, we examined the effects of H2S on [Ca2+]i in astrocytes from the rat spinal cord and whether H2S affects ATP-induced Ca2+ signaling, which is known to be involved in synaptic function. Na2S (150 μM), an H2S donor, produced a nontoxic increase in [Ca2+]i. The [Ca2+]i increase by Na2S was inhibited by Ca2+ depletion in the endoplasmic reticulum (ER) but not by removal of extracellular Ca2+, indicating that H2S releases Ca2+ from the ER. On the other hand, Na2S inhibited ATP-induced [Ca2+]i increase when Na2S clearly increased [Ca2+]i in the astrocytes, which was not suppressed by a reducing agent. In addition, Na2S had no effect on intracellular cyclic AMP (cAMP) level. These results indicate that oxidative post-translational modification of proteins and cAMP are not involved in the inhibitory effect of H2S on ATP-induced Ca2+ signaling. We conclude that H2S indirectly inhibits ATP-induced Ca2+ signaling by decreasing Ca2+ content in the ER in astrocytes. In this way, H2S may influence intercellular communication between astrocytes and neurons, thereby contributing to neuronal signaling in the nervous system.

G-Protein Subunit Gαi in Mitochondria, MrGPA1, Affects Conidiation, Stress Resistance, and Virulence of Entomopathogenic Fungus Metarhizium robertsii.

G proteins are critical modulators or transducers in various transmembrane signaling systems. They play key roles in numerous biological processes in fungi, including vegetative growth, development of infection-related structures, asexual conidiation, and virulence. However, functions of G proteins in entomopathogenic fungi remain unclear. Here, we characterized the roles of MrGPA1, a G-protein subunit Gαi, in conidiation, stress resistance, and virulence in Metarhizium robertsii. MrGPA1 was localized in the mitochondria. MrGpa1 deletion resulted in a significant reduction (47%) in the conidiation capacity, and reduced expression of several key conidiation-related genes, including fluG, flbD, brlA, wetA, phiA, and stuA. Further, MrGpa1 disruption resulted in decreased fungal sensitivity to UV irradiation and thermal stress, as determined based on conidial germination of ΔMrGpa1 and wild-type (WT) strains. Chemical stress analysis indicated that MrGpa1 contributes to fungal antioxidant capacity and cell wall integrity, but is not involved in tolerance to antifungal drug and osmotic stress. Importantly, insect bioassays involving (topical inoculation and injection) of Galleria mellonella larvae revealed decreased virulence of ΔMrGpa1 strain after cuticle infection. This was accompanied by decreased rates of appressorium formation and reduced expression of several cuticle penetration-related genes. Further assays showed that MrGpa1 regulated intracellular cyclic AMP (cAMP) levels, but feeding with cAMP could not recover the appressorium formation rate of ΔMrGpa1. These observations suggest that MrGpa1 contributes to the regulation of conidiation, UV irradiation, thermal stress response, antioxidant capacity, and cell wall integrity in M. robertsii. This gene is also involved in insect cuticle penetration during infection. These findings raise the possibility of designing powerful strategies for genetic improvement of M. robertsii conidiation capacity and virulence for killing pests.

A point mutation in the β-adrenergic-like octopamine receptor: possible association with amitraz resistance.

Amitraz is a unique formamidine-class acaricide/insecticide that effectively controls ticks, mites, and insect pests. However, the recent emergence of amitraz-resistant cattle ticks is a serious problem that requires an urgent solution. A nonsynonymous single nucleotide polymorphism (A181T) leading to an amino acid substitution (I61F) in the β-adrenergic-like (β-AL) octopamine receptor (OAR) of amitraz-resistant southern cattle ticks (Rhipicephalus microplus) (RmβAOR) was proposed to be a cause of the amitraz resistance. However, it remains unclear whether this substitution exerts any functional effect on the action of amitraz. To make this clear, the functional role of this mutation was examined using an orthologous OAR (BmOAR2) from the silkworm (Bombyx mori). Both amitraz and its metabolite N2 -(2,4-dimethylphenyl)-N1 -methyformamidine (DPMF) elevated intracellular cyclic AMP levels as orthosteric OAR agonists in HEK-293 cells stably expressing BmOAR2. The I45F mutant of BmOAR2 (equivalent to I61F in RmβAOR) was generated and tested for its sensitivity to amitraz and DPMF. The assay result showed that the I45F mutation reduces the potency of DPMF to a level similar to that of the endogenous agonist (R)-OA in wild-type BmOAR2. The amino acid substitution found in the first transmembrane segment of RmβAOR most likely causes target-site insensitivity to DPMF, which might contribute to the resistance of R. microplus to amitraz. This needs to be further confirmed using RmβAOR. © 2020 Society of Chemical Industry.

Abstract A10: The dual A2aR/A2bR antagonist AB928 reverses adenosine-mediated immune suppression and inhibits tumor growth in vivo

Abstract Introduction: High levels of immunosuppressive adenosine are found in the tumor microenvironment, reaching 50-100 μM in experimental models. Adenosine exerts its effects on immune cells primarily through the adenosine receptors A2aR/A2bR, which increase intracellular levels of cyclic AMP, leading to CREB phosphorylation (pCREB). We have previously shown that the dual A2aR/A2bR antagonist AB928 is capable of inhibiting adenosine-induced pCREB in healthy human volunteer (HV) blood lymphocytes. AB928 has also been shown to relieve adenosine-mediated T-cell suppression in vitro and exhibit combinatorial effects with standard-of-care chemotherapeutics in mouse syngeneic tumor models. Herein, we show that AB928 is capable of inhibiting NECA-induced gene expression changes and CREB phosphorylation in non-small cell lung carcinoma (NSCLC) patient whole blood (WB). Additionally, observations from our in vitro human studies showing the combinatorial effect of AB928 and α-PD-1 were reproduced in B16F10 syngeneic tumors. Methods: Human WB was stimulated with 5 μM of NECA and flow cytometry was used to quantify AB928-mediated inhibition of pCREB and CD3ζ phosphorylation. B16F10 tumors were treated with α-PD-1 +/- AB928 and gene expression was determined from excised mouse tumors using the nCounter PanCancer panel. Results: To ensure AB928 can successfully inhibit the high levels of intratumoral adenosine, we found that 5 μM NECA provides maximal stimulation and is significantly more potent (>20 fold) than adenosine in the pCREB assay. Additional experiments demonstrated that AB928 has comparable potency in NECA-stimulated WB from both HV and NSCLC patients. In addition to blocking downstream signaling, NanoString analysis showed that AB928 could prevent NECA-stimulated gene expression changes in NSCLC WB. We also found that NECA stimulation, alone or in combination with PD-1 inhibition, significantly reduced proximal TCR signaling, leading to reduced levels of CD3ζ phosphorylation at TYR142 (pTYR142). These reduced pTYR142 levels, with and without α-PD-1, could be significantly rescued by AB928, suggesting that blocking adenosine immunosuppression may provide additional benefit to PD-1 inhibition in tumors. Consistent with these results, AB928 was capable of suppressing growth (volume in mm3) of B16-F10 tumors both as a single agent (vehicle: 462 +/- 58; AB928: 292 +/- 55; p<0.05) or in combination with α-PD-1 therapy (α-PD-1: 341 +/- 60; AB928+α-PD-1: 123 +/- 30; p<0.05). Further, NanoString analysis of the tumors showed an increased “T-cell Functions” score with both α-PD-1 and AB928 monotherapy, which was further enhanced in combination treatment. Conclusions: Collectively, these results support a role for AB928 in relieving adenosine-mediated immunosuppression by blocking A2aR/A2bR-induced signaling events, gene expression changes, and suppressing tumor growth in vivo. Citation Format: Daniel M. DiRenzo, Nell Narasappa, Dana Piovesan, Devika Ashok, Park Adam, Jenna L. Jeffrey, Manmohan R. Leleti, Joanne B.L. Tan, Lisa Seitz, Steve W. Young, Jay P. Powers, Matthew J. Walters. The dual A2aR/A2bR antagonist AB928 reverses adenosine-mediated immune suppression and inhibits tumor growth in vivo [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A10.

Post-ejaculatory modifications to sperm (PEMS).

Mammalian sperm must spend a minimum period of time within a female reproductive tract to achieve the capacity to fertilize oocytes. This phenomenon, termed sperm 'capacitation', was discovered nearly seven decades ago and opened a window into the complexities of sperm-female interaction. Capacitation is most commonly used to refer to a specific combination of processes that are believed to be widespread in mammals and includes modifications to the sperm plasma membrane, elevation of intracellular cyclic AMP levels, induction of protein tyrosine phosphorylation, increased intracellular Ca2+ levels, hyperactivation of motility, and, eventually, the acrosome reaction. Capacitation is only one example of post-ejaculatory modifications to sperm (PEMS) that are widespread throughout the animal kingdom. Although PEMS are less well studied in non-mammalian taxa, they likely represent the rule rather than the exception in species with internal fertilization. These PEMS are diverse in form and collectively represent the outcome of selection fashioning complex maturational trajectories of sperm that include multiple, sequential phenotypes that are specialized for stage-specific functionality within the female. In many cases, PEMS are critical for sperm to migrate successfully through the female reproductive tract, survive a protracted period of storage, reach the site of fertilization and/or achieve the capacity to fertilize eggs. We predict that PEMS will exhibit widespread phenotypic plasticity mediated by sperm-female interactions. The successful execution of PEMS thus has important implications for variation in fitness and the operation of post-copulatory sexual selection. Furthermore, it may provide a widespread mechanism of reproductive isolation and the maintenance of species boundaries. Despite their possible ubiquity and importance, the investigation of PEMS has been largely descriptive, lacking any phylogenetic consideration with regard to divergence, and there have been no theoretical or empirical investigations of their evolutionary significance. Here, we (i) clarify PEMS-related nomenclature; (ii) address the evolutionary origin, maintenance and divergence in PEMS in the context of the protracted life history of sperm and the complex, selective environment of the female reproductive tract; (iii) describe taxonomically widespread types of PEMS: sperm activation, chemotaxis and the dissociation of sperm conjugates; (iv) review the occurence of PEMS throughout the animal kingdom; (v) consider alternative hypotheses for the adaptive value of PEMS; (vi) speculate on the evolutionary implications of PEMS for genomic architecture, sexual selection, and reproductive isolation; and (vii) suggest fruitful directions for future functional and evolutionary analyses of PEMS.

Intracellular cyclic AMP levels modulate differential adaptive responses on epimastigotes and cell culture trypomastigotes of Trypanosoma cruzi

Among the many environmental challenges the parasite Trypanosoma cruzi has to overcome to complete its life cycle through different hosts, oxidative stress plays a central role. Different stages of this parasite encounter distinct sources of oxidative stress, such as the oxidative burst of the immune system, or the Heme released from hemoglobin degradation in the triatomine's midgut. Also, the redox status of the surroundings functions as a signal to the parasite, triggering processes coupled to differentiation or proliferation. Intracellular second messengers, like cAMP, are responsible for the transduction of environmental queues and initiating cellular processes accordingly. In trypanosomatids cAMP is involved in a variety of processes, including proliferation, differentiation, osmoregulation and quorum sensing. Trypanosomatid phosphodiesterases (PDE) show atypical pharmacological properties and some have been involved in key processes for the survival of the parasites, which validates them as attractive therapeutic targets. Our work here shows that cAMP modulates different processes according to parasite stage. Epimastigotes become more resistant to oxidative stress when pre-treated with cAMP analogs, while in trypomastigotes an increase in intracellular cAMP doesn't seem to aid in this response, although it does increase the number of amastigotes obtained 48 h after infection, compared to the control group. Also, we show that TcrPDEA1, a functionally enigmatic phosphodiesterase with very high Km, is involved in the epimastigotes response to oxidative stress.

Alpha 2 adrenoceptor agonist guanabenz directly inhibits hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels in mesencephalic trigeminal nucleus neurons

Alpha 2 (α2-) adrenoceptor agonists, such as clonidine or dexmedetomidine, have been found to inhibit hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channels, not only by reducing intracellular cyclic AMP levels but also by directly blocking HCN channels. In this study, we examined the inhibitory effect of guanabenz, a centrally acting α2-adrenoceptor agonist with high specificity for α2A-subtype, on HCN channels in mesencephalic trigeminal nucleus (MTN) neurons which robustly express HCN channels and have been suggested to coexpress α2A-adrenoceptors. By performing whole-cell patch-clamp recording on MTN neurons in brainstem slices, hyperpolarization-activated inward current (Ih) was examined during guanabenz treatment. Guanabenz inhibited Ih in a dose-dependent manner, which was likely to be ZD7288-sensitive HCN current as it did not affect barium-sensitive inward rectifying potassium current. Guanabenz not only inhibited Ih but also shifted the voltage-dependent activation curve to hyperpolarizing potentials. Interestingly, Ih inhibition by guanabenz was not reversed by α2-adrenoceptor antagonist atipamezole treatment or by intracellular cyclic AMP perfusion, suggesting that the inhibition may not result from α2A-adrenoceptor signalling pathway but from direct inhibition of HCN channels. Coherent to our electrophysiological results, single-cell RT-PCR revealed that most MTN neurons lack α2A-adrenoceptor mRNA. Our study demonstrates that guanabenz can directly inhibit HCN channels in addition to its primary role of activating α2A-adrenoceptors.

Involvement of the Adhesion GPCRs Latrophilins in the Regulation of Insulin Release

Insulin secretion from pancreatic β cells is a highly complex and tightly regulated process. Its dysregulation is one characteristic of type 2 diabetes, and thus, an in-depth understanding of the mechanisms controlling insulin secretion is essential for rational therapeutic intervention. G-protein-coupled receptors (GPCRs) have been established as major regulators of insulin exocytosis. Recent studies also suggest the involvement of adhesion GPCRs, a non-prototypical class of GPCRs. Here, we identify latrophilins, which belong to the class of adhesion GPCRs, to be highly expressed in different cell types of pancreatic islets. Invitro and exvivo analyses show that distinct splice variants of the latrophilin LPHN3/ADGRL3 decrease insulin secretion from pancreatic β cells by reducing intracellular cyclic AMP levels via the Gi-mediated pathway. Our data highlight the key role of LPHN3 in modulating insulin secretion and its potential as therapeutic target for type 2 diabetes.

Phosphodiesterase 4B is an effective therapeutic target in colorectal cancer

Identification of new therapeutic targets may improve the survival rate of patients with colorectal cancer (CRC). Recent studies have suggested that the level of phosphodiesterase 4B (PDE4B) is elevated in fatal/refractory diffuse large B-cell lymphoma (DLBCL), and therapeutic efficacy of a PDE4 inhibitor in B-cell lymphoma has been successfully tested in clinical settings. Here, we show that PDE4B is a potential therapeutic target in CRC. Treatment with forskolin, an activator of adenylyl cyclase (AC), increased intracellular cyclic AMP (cAMP) levels in PDE4B-low, but not PDE4B-high cells, indicating that PDE4B was a major regulator of cAMP levels in CRC cells. Furthermore, cAMP modulated the activities of AKT and AMPK in a PDE4B-dependent manner, which was associated with a marked decrease in mTOR-Myc signals and oncogenic properties of CRC cells such as anchorage-independent growth and colony formation. We found that the Myc proto-oncogene was a crucial downstream target of the AKT/mTOR and AMPK/mTOR signals that mediated cAMP-induced anti-tumor effect. A natural polyphenol resveratrol that was reported to have PDE4 inhibitory effects also showed tumor suppressive effects by inhibiting the mTOR-Myc axis. Intriguingly, we identified Myc as a transcriptional activator of PDE4B in CRC cells, which maintains the intracellular cAMP levels low and promotes cell survival. These data suggest that cAMP/PDE4B signals play a significant role in regulating the malignant phenotype of CRC cells and targeting of PDE4B should be actively pursued.

Control of nongenetic heterogeneity in growth rate and stress tolerance of Saccharomyces cerevisiae by cyclic AMP-regulated transcription factors.

Genetically identical cells exhibit extensive phenotypic variation even under constant and benign conditions. This so-called nongenetic heterogeneity has important clinical implications: within tumors and microbial infections, cells show nongenetic heterogeneity in growth rate and in susceptibility to drugs or stress. The budding yeast, Saccharomyces cerevisiae, shows a similar form of nongenetic heterogeneity in which growth rate correlates positively with susceptibility to acute heat stress at the single-cell level. Using genetic and chemical perturbations, combined with high-throughput single-cell assays of yeast growth and gene expression, we show here that heterogeneity in intracellular cyclic AMP (cAMP) levels acting through the conserved Ras/cAMP/protein kinase A (PKA) pathway and its target transcription factors, Msn2 and Msn4, underlies this nongenetic heterogeneity. Lower levels of cAMP correspond to slower growth, as shown by direct comparison of cAMP concentration in subpopulations enriched for slower vs. faster growing cells. Concordantly, an endogenous reporter of this pathway’s activity correlates with growth in individual cells. The paralogs Msn2 and Msn4 differ in their roles in nongenetic heterogeneity in a way that demonstrates slow growth and stress tolerance are not inevitably linked. Heterogeneity in growth rate requires each, whereas only Msn2 is required for heterogeneity in expression of Tsl1, a subunit of trehalose synthase that contributes to acute-stress tolerance. Perturbing nongenetic heterogeneity by mutating genes in this pathway, or by culturing wild-type cells with the cell-permeable cAMP analog 8-bromo-cAMP or the PKA inhibitor H89, significantly impacts survival of acute heat stress. Perturbations that increase intracellular cAMP levels reduce the slower-growing subpopulation and increase susceptibility to acute heat stress, whereas PKA inhibition slows growth and decreases susceptibility to acute heat stress. Loss of Msn2 reduces, but does not completely eliminate, the correlation in individual cells between growth rate and acute-stress survival, suggesting a major role for the Msn2 pathway in nongenetic heterogeneity but also a residual benefit of slow growth. Our results shed light on the genetic control of nongenetic heterogeneity and suggest a possible means of defeating bet-hedging pathogens or tumor cells by making them more uniformly susceptible to treatment.