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Adenylyl cyclase isoform 1 (AC1) is considered a promising target for treating inflammatory pain. Our group identified the pyrazolyl-pyrimidinone scaffold as potent and selective inhibitors of Ca 2+ /CaM-mediated AC1 activity; however, the molecules suffered from poor aqueous solubility. The current study presents a strategy to improve aqueous solubility of the scaffold by reduction of crystal packing energy and increasing rotational degrees of freedom within the molecule. Structure-activity and property relationship studies identified the second generation lead 7-47A ( AC10142A ) that demonstrated and AC1 IC 50 value of 0.26 μM and aqueous solubility of 74 ± 7 μM. After in vitro ADME characterization, the scaffold advanced to in vivo pharmacokinetic evaluation, demonstrating adequate levels of exposure. Finally, 7-47A exhibited antiallodynic efficacy in a rat complete Freund's adjuvant model for inflammatory pain showing improvement over previous iterations of this scaffold. These results further validate AC1 inhibition as a viable therapeutic strategy for treating chronic and inflammatory pain.

Globally, despite extensive research and pharmacological advancement, cancer remains one of the most common causes of mortality. Understanding the signaling pathways involved in cancer progression is essential for the discovery of new drug targets. The adenylyl cyclase (AC) superfamily comprises glycoproteins that regulate intracellular signaling and convert ATP into cyclic AMP, an important second messenger. The present review highlights the involvement of ACs in cancer progression and suppression, broken down for each specific mammalian AC isoform. The precise mechanisms by which ACs contribute to cancer cell proliferation and invasion are not well understood and are variable among cancer types; however, AC overactivation, along with that of downstream regulators, presents a potential target for novel anticancer therapies. The expression patterns of ACs in numerous cancers are discussed. In addition, we highlight inhibitors of AC-related signaling that are currently under investigation, with a focus on possible anti-cancer strategies. Recent discoveries with small molecules regarding more direct modulation AC activity are also discussed in detail. A more comprehensive understanding of different components in AC-related signaling could potentially lead to the development of novel therapeutic strategies for personalized oncology and might enhance the efficacy of chemoimmunotherapy in the treatment of various cancers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Pain and anxiety are two common and undertreated non-motor symptoms in Parkinson's disease (PD), which affect the life quality of PD patients, and the underlying mechanisms remain unclear. As an important subtype of adenylyl cyclases (ACs), adenylyl cyclase subtype 1 (AC1) is critical for the induction of cortical long-term potentiation (LTP) and injury induced synaptic potentiation in the cortical areas including anterior cingulate cortex (ACC) and insular cortex (IC). Genetic deletion of AC1 or pharmacological inhibition of AC1 improved chronic pain and anxiety in different animal models. In this study, we proved the motor deficit, pain and anxiety symptoms of PD in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice model. As a lead candidate AC1 inhibitor, oral administration (1 dose and seven doses) of NB001 (20 and 40 mg/kg) showed significant analgesic effect in MPTP-treated mice, and the anxiety behavior was also reduced (40 mg/kg). By using genetic knockout mice, we found that AC1 knockout mice showed reduced pain and anxiety symptoms after MPTP administration, but not AC8 knockout mice. In summary, genetic deletion of AC1 or pharmacological inhibition of AC1 improved pain and anxiety symptoms in PD model mice, but didn't affect motor function. These results suggest that NB001 is a potential drug for the treatment of pain and anxiety symptoms in PD patients by inhibiting AC1 target., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Nearly half of all pregnancies are unintended; thus, existing family planning options are inadequate. For men, the only choices are condoms and vasectomy, and most current efforts to develop new contraceptives for men impact sperm development, meaning that contraception requires months of continuous pretreatment. Here, we provide proof-of-concept for an innovative strategy for on-demand contraception, where a man would take a birth control pill shortly before sex, only as needed. Soluble adenylyl cyclase (sAC) is essential for sperm motility and maturation. We show a single dose of a safe, acutely-acting sAC inhibitor with long residence time renders male mice temporarily infertile. Mice exhibit normal mating behavior, and full fertility returns the next day. These studies define sAC inhibitors as leads for on-demand contraceptives for men, and they provide in vivo proof-of-concept for previously untested paradigms in contraception; on-demand contraception after just a single dose and pharmacological contraception for men., (© 2023. The Author(s).)

Osteocytes respond to mechanical forces controlling osteoblast and osteoclast function. Mechanical stimulation decreases osteocyte apoptosis and promotes bone formation. Primary cilia have been described as potential mechanosensors in bone cells. Certain osteogenic responses induced by fluid flow (FF) in vitro are decreased by primary cilia inhibition in MLO-Y4 osteocytes. The parathyroid hormone (PTH) receptor type 1 (PTH1R) modulates osteoblast, osteoclast, and osteocyte effects upon activation by PTH or PTH-related protein (PTHrP) in osteoblastic cells. Moreover, some actions of PTH1R seem to be triggered directly by mechanical stimulation. We hypothesize that PTH1R forms a signaling complex in the primary cilium that is essential for mechanotransduction in osteocytes and affects osteocyte-osteoclast communication. MLO-Y4 osteocytes were stimulated by FF or PTHrP (1-37). PTH1R and primary cilia signaling were abrogated using PTH1R or primary cilia specific siRNAs or inhibitors, respectively. Conditioned media obtained from mechanically- or PTHrP-stimulated MLO-Y4 cells inhibited the migration of preosteoclastic cells and osteoclast differentiation. Redistribution of PTH1R along the entire cilium was observed in mechanically stimulated MLO-Y4 osteocytic cells. Preincubation of MLO-Y4 cells with the Gli-1 antagonist, the adenylate cyclase inhibitor (SQ22536), or with the phospholipase C inhibitor (U73122), affected the migration of osteoclast precursors and osteoclastogenesis. Proteomic analysis and neutralizing experiments showed that FF and PTH1R activation control osteoclast function through the modulation of C-X-C Motif Chemokine Ligand 5 (CXCL5) and interleukin-6 (IL-6) secretion in osteocytes. These novel findings indicate that both primary cilium and PTH1R are necessary in osteocytes for proper communication with osteoclasts and show that mechanical stimulation inhibits osteoclast recruitment and differentiation through CXCL5, while PTH1R activation regulate these processes via IL-6., (© 2022 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

A series of acyclic nucleoside phosphonates (ANPs) was designed as inhibitors of bacterial adenylate cyclases (ACs), where adenine was replaced with 2-amino-4-arylthiazoles. The target compounds were prepared using the halogen dance reaction. Final AC inhibitors were evaluated in cell-based assays (prodrugs) and cell-free assays (phosphono diphosphates). Novel ANPs were potent inhibitors of adenylate cyclase toxin (ACT) from Bordetella pertussis and edema factor (EF) from Bacillus anthracis, with substantial selectivity over mammalian enzymes AC1, AC2, and AC5. Six of the new ANPs were more potent or equipotent ACT inhibitors (IC 50 =9-18 nM), and one of them was more potent EF inhibitor (IC 50 =12 nM), compared to adefovir diphosphate (PMEApp) with IC 50 =18 nM for ACT and IC 50 =36 nM for EF. Thus, these compounds represent the most potent ACT/EF inhibitors based on ANPs reported to date. The potency of the phosphonodiamidates to inhibit ACT activity in J774A.1 macrophage cells was somewhat weaker, where the most potent derivative had IC 50 =490 nM compared to IC 50 =150 nM of the analogous adefovir phosphonodiamidate. The results suggest that more efficient type of phosphonate prodrugs would be desirable to increase concentrations of the ANP-based active species in the cells in order to proceed with the development of ANPs as potential antitoxin therapeutics., (© 2021 Wiley-VCH GmbH.)

Mobilization of astrocyte glycogen is key for processes such as synaptic plasticity and memory formation but the link between neuronal activity and glycogen breakdown is not fully known. Activation of cytosolic soluble adenylyl cyclase (sAC) in astrocytes has been suggested to link neuronal depolarization and glycogen breakdown partly based on experiments employing pharmacological inhibition of sAC. However, several studies have revealed that sAC located within mitochondria is a central regulator of respiration and oxidative phosphorylation. Thus, pharmacological sAC inhibition is likely to affect both cytosolic and mitochondrial sAC and if bioenergetic readouts are studied, the observed effects are likely to stem from inhibition of mitochondrial rather than cytosolic sAC. Here, we report that a pharmacologically induced inhibition of sAC activity lowers mitochondrial respiration, induces phosphorylation of the metabolic master switch AMP-activated protein kinase (AMPK), and decreases glycogen stores in cultured primary murine astrocytes. From these data and our discussion of the literature, mitochondrial sAC emerges as a key regulator of astrocyte bioenergetics. Lastly, we discuss the challenges of investigating the functional and metabolic roles of cytosolic versus mitochondrial sAC in astrocytes employing the currently available pharmacological tool compounds., (© 2021 Wiley Periodicals LLC.)

A series of novel acyclic nucleoside phosphonates (ANPs) was synthesized as potential adenylate cyclase inhibitors, where the adenine nucleobase of adefovir (PMEA) was replaced with a 5-substituted 2-aminothiazole moiety. The design was based on the structure of MB05032, a potent and selective inhibitor of fructose 1,6-bisphosphatase and a good mimic of adenosine monophosphate (AMP). From the series of eighteen novel ANPs, which were prepared as phosphoroamidate prodrugs, fourteen compounds were potent (single digit micromolar or submicromolar) inhibitors of Bordetella pertussis adenylate cyclase toxin (ACT), mostly without observed cytotoxicity in J774A.1 macrophage cells. Selected phosphono diphosphates (nucleoside triphosphate analogues) were potent inhibitors of ACT (IC 50 as low as 37 nM) and B. anthracis edema factor (IC 50 as low as 235 nM) in enzymatic assays. Furthermore, several ANPs were found to be selective mammalian AC1 inhibitors in HEK293 cell-based assays (although with some associated cytotoxicity) and one compound exhibited selective inhibition of mammalian AC2 (only 12% of remaining adenylate cyclase activity) but no observed cytotoxicity. The mammalian AC1 inhibitors may represent potential leads in development of agents for treatment of human inflammatory and neuropathic pain., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

The acidic tumor microenvironment in melanoma drives immune evasion by up-regulating cyclic adenosine monophosphate (cAMP) in tumor-infiltrating monocytes. Here we show that the release of non-toxic concentrations of an adenylate cyclase (AC) inhibitor from poly(sarcosine)-block-poly(L-glutamic acid γ-benzyl ester) (polypept(o)id) copolymer micelles restores antitumor immunity. In combination with selective, non-therapeutic regulatory T cell depletion, AC inhibitor micelles achieve a complete remission of established B16-F10-OVA tumors. Single-cell sequencing of melanoma-infiltrating immune cells shows that AC inhibitor micelles reduce the number of anti-inflammatory myeloid cells and checkpoint receptor expression on T cells. AC inhibitor micelles thus represent an immunotherapeutic measure to counteract melanoma immune escape., (© 2021. The Author(s).)

Soluble adenylyl cyclase (sAC: ADCY10) has been genetically confirmed to be essential for male fertility in mice and humans. In mice, ex vivo studies of dormant, caudal epididymal sperm demonstrated that sAC is required for initiating capacitation and activating motility. We now use an improved sAC inhibitor, TDI-10229, for a comprehensive analysis of sAC function in mouse and human sperm. In contrast to caudal epididymal mouse sperm, human sperm are collected post-ejaculation, after sAC activity has already been stimulated. In addition to preventing the capacitation-induced stimulation of sAC and protein kinase A activities, tyrosine phosphorylation, alkalinization, beat frequency and acrosome reaction in dormant mouse sperm, sAC inhibitors interrupt each of these capacitation-induced changes in ejaculated human sperm. Furthermore, we show for the first time that sAC is required during acrosomal exocytosis in mouse and human sperm. These data define sAC inhibitors as candidates for non-hormonal, on-demand contraceptives suitable for delivery via intravaginal devices in women., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

[Figure: see text].

Bacillus anthracis edema toxin (ET) inhibited lethal toxin-stimulated pulmonary artery pressure (Ppa) and increased lung cAMP levels in our previous study. We therefore examined whether ET inhibits hypoxic pulmonary vasoconstriction (HPV). Following baseline hypoxic measures in isolated perfused lungs from healthy rats, compared with diluent, ET perfusion reduced maximal Ppa increases (mean ± SE percentage of maximal Ppa increase with baseline hypoxia) during 6-min hypoxic periods (FIO 2  = 0%) at 120 min (16 ± 6% vs. 51 ± 6%, P = 0.004) and 180 min (11.4% vs. 55 ± 6%, P = 0.01). Protective antigen-mAb (PA-mAb) and adefovir inhibit host cell edema factor uptake and cAMP production, respectively. In lungs perfused with ET following baseline measures, compared with placebo, PA-mAb treatment increased Ppa during hypoxia at 120 and 180 min (56 ± 6% vs. 10 ± 4% and 72 ± 12% vs. 12 ± 3%, respectively, P ≤ 0.01) as did adefovir (84 ± 10% vs. 16.8% and 123 ± 21% vs. 26 ± 11%, respectively, P ≤ 0.01). Compared with diluent, lung perfusion with ET for 180 min reduced the slope of the relationships between Ppa and increasing concentrations of endothelin-1 (ET-1) (21.12 ± 2.96 vs. 3.00 ± 0.76 × 10 8 cmH 2 O/M, P < 0.0001) and U46619, a thromboxane A2 analogue (7.15 ± 1.01 vs. 3.74 ± 0.31 × 10 7 cmH 2 O/M, P = 0.05) added to perfusate. In lungs isolated from rats after 15 h of in vivo infusions with either diluent, ET alone, or ET with PA-mAb, compared with diluent, the maximal Ppa during hypoxia and the slope of the relationship between change in Ppa and ET-1 concentration added to the perfusate were reduced in lungs from animals challenged with ET alone ( P ≤ 0.004) but not with ET and PA-mAb together ( P ≥ 0.73). Inhibition of HPV by ET could aggravate hypoxia during anthrax pulmonary infection. NEW & NOTEWORTHY The most important findings here are edema toxin's potent adenyl cyclase activity can interfere with hypoxic pulmonary vasoconstriction, an action that could worsen hypoxemia during invasive anthrax infection with lung involvement. These findings, coupled with other studies showing that lethal toxin can disrupt pulmonary vascular integrity, indicate that both toxins can contribute to pulmonary pathophysiology during infection. In combination, these investigations provide a further basis for the use of antitoxin therapies in patients with worsening invasive anthrax disease.

Hepatic ischemia/reperfusion (I/R) injury is a leading cause of organ dysfunction and failure in numerous pathological and surgical settings. At the core of this issue lies mitochondrial dysfunction. Hence, strategies that prime mitochondria towards damage resilience might prove applicable in a clinical setting. A promising approach has been to induce a mitohormetic response, removing less capable organelles, and replacing them with more competent ones, in preparation for an insult. Recently, a soluble form of adenylyl cyclase (sAC) has been shown to exist within mitochondria, the activation of which improved mitochondrial function. Here, we sought to understand if inhibiting mitochondrial sAC would elicit mitohormesis and protect the liver from I/R injury. Wistar male rats were pretreated with LRE1, a specific sAC inhibitor, prior to the induction of hepatic I/R injury, after which mitochondria were collected and their metabolic function was assessed. We find LRE1 to be an effective inducer of a mitohormetic response based on all parameters tested, a phenomenon that appears to require the activity of the NAD + -dependent sirtuin deacylase (SirT3) and the subsequent deacetylation of mitochondrial proteins. We conclude that LRE1 pretreatment leads to a mitohormetic response that protects mitochondrial function during I/R injury.

Coral calcification is intricately linked to the chemical composition of the fluid in the extracellular calcifying medium (ECM), which is situated between the calcifying cells and the skeleton. Here we demonstrate that the acid-base sensing enzyme soluble adenylyl cyclase (sAC) is expressed in calcifying cells of the coral Stylophora pistillata. Furthermore, pharmacological inhibition of sAC in coral microcolonies resulted in acidification of the ECM as estimated by the pH-sensitive ratiometric indicator SNARF, and decreased calcification rates, as estimated by calcein labeling of crystal growth. These results indicate that sAC activity modulates some of the molecular machinery involved in producing the coral skeleton, which could include ion-transporting proteins and vesicular transport. To our knowledge this is the first study to directly demonstrate biological regulation of the alkaline pH of the coral ECM and its correlation with calcification., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Heterologous sensitization of adenylyl cyclase (AC) is revealed as enhanced or exaggerated AC/cAMP signaling that occurs following persistent activation of G α i/o -coupled receptors. This paradoxical phenomenon was discovered more than 40 years ago and was proposed as a cellular mechanism to explain the adaptive changes that occur following chronic exposure to drugs of abuse. However, the underlying molecular mechanisms of heterologous sensitization of AC remain largely unknown. In the present study, we performed a genome-wide cell-based RNA interference screen as an unbiased approach to identify genes associated with heterologous sensitization of AC. Following a series of validation and confirmation assays, three genes that form an E3 ligase complex, cullin3 ( CUL3 ), neural precursor-cell-expressed and developmentally downregulated 8 ( NEDD8 ), and really interesting new gene (RING)-box protein 1 ( RBX1 ), were identified as specific modulators of heterologous sensitization of AC. Furthermore, based on the downstream actions of these genes, we evaluated the activity of proteasome inhibitors as well as the specific NEDD8-activating enzyme inhibitor, MLN4924 (Pevonedistat), in AC sensitization. We demonstrate that MG-132 and bortezomib treatments could mimic the inhibitory effects observed with gene knockdown, and MLN4924 was potent and efficacious in blocking the development of heterologous sensitization of endogenous and recombinant AC isoforms, including AC1, AC2, AC5, and AC6. Together, by using genetic and pharmacological approaches, we identified, for the first time, cullin3-RING ligases and the protein degradation pathway as essential modulators for heterologous sensitization of AC. SIGNIFICANCE STATEMENT: Through a genome-wide cell-based RNA interference screening, we identified three genes that form an E3 ligase complex, cullin3, neural precursor-cell-expressed and developmentally downregulated 8 ( NEDD8 ), and really interesting new gene-box protein 1, as specific modulators of heterologous sensitization of AC. The effect of cullin3 , NEDD8, or really interesting new gene-box protein 1 small interfering RNAs on heterologous sensitization was recapitulated by proteasome inhibitors, MG132 and bortezomib, and the specific NEDD8-activating enzyme inhibitor, MLN4924. These results suggest a novel hypothesis in which protein degradation is involved in the sensitization of AC signaling that occurs following chronic activation of Gα i/o -coupled receptors., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

The soluble adenylyl cyclase (sAC) was identified in the heart as another source of cyclic AMP (cAMP). However, its cardiac physiological function is unknown. On the other hand, the cardiac Na + /HCO 3 - cotransporter (NBC) promotes the cellular co-influx of HCO 3 - and Na + . Since sAC activity is regulated by HCO 3 - , our purpose was to investigate the potential functional relationship between NBC and sAC in the cardiomyocyte. Rat ventricular myocytes were loaded with Fura-2, Fluo-3, or BCECF to measure Ca 2+ transient (Ca 2+ i ) by epifluorescence, Ca 2+ sparks frequency (CaSF) by confocal microscopy, or intracellular pH (pH i ) by epifluorescence, respectively. Sarcomere or cell shortening was measured with a video camera as an index of contractility. The NBC blocker S0859 (10 μM), the selective inhibitor of sAC KH7 (1 μM), and the PKA inhibitor H89 (0.1 μM) induced a negative inotropic effect which was associated with a decrease in Ca 2+ i . Since PKA increases Ca 2+ release through sarcoplasmic reticulum RyR channels, CaSF was measured as an index of RyR open probability. The generation of CaSF was prevented by KH7. Finally, we investigated the potential role of sAC activation on NBC activity. NBC-mediated recovery from acidosis was faster in the presence of KH7 or H89, suggesting that the pathway sAC-PKA is negatively regulating NBC function, consistent with a negative feedback modulation of the HCO 3 - influx that activates sAC. In summary, the results demonstrated that the complex NBC-sAC-PKA plays a relevant role in Ca 2+ handling and basal cardiac contractility.

Literature studies suggest important protective effects of low-frequency, low-energy pulsed electromagnetic fields (PEMFs) on inflammatory pathways affecting joint and cerebral diseases. However, it is not clear on which bases they affect neuroprotection and the mechanism responsible is yet unknown. Therefore the aim of this study was to identify the molecular targets of PEMFs anti-neuroinflammatory action. The effects of PEMF exposure in cytokine production by lipopolysaccharide (LPS)-activated N9 microglial cells as well as the pathways involved, including adenylyl cyclase (AC), phospholipase C (PLC), protein kinase C epsilon (PKC-ε) and delta (PKC-δ), p38, ERK1/2, JNK1/2 mitogen activated protein kinases (MAPK), Akt and caspase 1, were investigated. In addition, the ability of PEMFs to modulate ROS generation, cell invasion and phagocytosis, was addressed. PEMFs reduced the LPS-increased production of TNF-α and IL-1β in N9 cells, through a pathway involving JNK1/2. Furthermore, they decreased the LPS-induced release of IL-6, by a mechanism not dependent on AC, PLC, PKC-ε, PKC-δ, p38, ERK1/2, JNK1/2, Akt and caspase 1. Importantly, a significant effect of PEMFs in the reduction of crucial cell functions specific of microglia like ROS generation, cell invasion and phagocytosis was found. PEMFs inhibit neuroinflammation in N9 cells through a mechanism involving, at least in part, the activation of JNK MAPK signalling pathway and may be relevant to treat a variety of diseases characterized by neuroinflammation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

We studied the involvement of cAMP/PKA signaling in the realization of the growth potential of neural progenitors and secretion of neurotrophic growth factors by glial elements under conditions of ethanol-induced neurodegeneration in vitro and in vivo. The stimulating role of cAMP and PKA in cell cycle progression of the neural progenitor cells and in production of neurotrophins by the cells in nervous tissue under the optimal conditions to vital activity was demonstrated. Ethanol inverted the role of cAMP/PKA signaling pathways in determination of the proliferation-differentiation status of neural stem cells. Selective blockade of adenylate cyclase or PKA in neural stem cells increased the rate of their division against the background of relative decrease in differentiation rate. In addition, cAMP/PKA signaling does not longer participate in neurotrophin production by glial cells in neurodegeneration. These findings suggest that inhibitors of activity/expression of adenylate cyclase and PKA can be considered as possible drugs with regenerative activity for the treatment of nervous system pathologies provoked by alcohol.

Response to stressors in our environment and daily lives is an adaptation conserved through evolution as it is beneficial in enhancing the survival and continuity of humans. Although stressors have evolved, the drastic physiological response they elicit still remains unchanged. The chronic secretion and circulation of catecholamines to produce physical responses when they are not required may result in pathological consequences which affect cardiac function drastically. This review seeks to point out the probable implication of chronic stress in inducing an inflammation disorder in the heart. We discussed the likely synergy of a G protein-independent stimuli signaling via β 2 -adrenergic receptors in both cardiomyocytes and immune cells during chronic catecholamine stress. To explain this synergy, we hypothesized the possibility of adenylyl cyclases having a regulatory effect on G protein-coupled receptor kinases. This was based on the negative correlations they exhibit during normal cardiac function and heart failures. As such, the downregulation of adenylyl cyclases in cardiomyocytes and immune cells during chronic catecholamine stress enhances the expressions of G protein-coupled receptor kinases. In addition, we explain the maladaptive roles played by G protein-coupled receptor kinase and extracellular signal-regulated kinase in the synergistic cascade that pathologically remodels the heart. Finally, we highlighted the therapeutic potentials of an adenylyl cyclases stimulator to attenuate pathological cardiac hypertrophy (PCH) and improve cardiac function in patients developing cardiac disorders due to chronic catecholamine stress.

Resveratrol (RSV) is a natural polyphenolic antioxidant with a proven protective role in several human diseases involving oxidative stress, although the molecular mechanism underlying this effect remains unclear. The present work tried to elucidate the molecular mechanism of RSV's role on signal transduction modulation. Our biochemical analysis, including radioligand binding, real time PCR, western blotting and adenylyl cyclase activity, and computational studies provide insights into the RSV binding pathway, kinetics and the most favored binding pose involving adenosine receptors, mainly A 2A subtype. In this study, we show that RSV target adenosine receptors (AdoRs), affecting gene expression, receptor levels, and the downstream adenylyl cyclase (AC)/PKA pathway. Our data demonstrate that RSV activates AdoRs. Moreover, RSV activate A 2A receptors by directly binding to the classical orthosteric binding site. Intriguingly, RSV-induced receptor activation can stimulate or inhibit AC activity depending on concentration and exposure time. Such subtle and multifaceted regulation of the AdoRs/AC/PKA pathway might contribute to the protective role of RSV. Our findings suggest that RSV molecular action is mediated, at least in part, by activation of adenosine receptors and create the opportunity to interrogate the therapeutic use of RSV in pathological conditions involving AdoRs, such as Alzheimer., (Copyright © 2019. Published by Elsevier Inc.)

Adenylyl cyclases are key points for the integration of stimulatory and inhibitory G protein-coupled receptor (GPCR) signals. Adenylyl cyclase type 5 (AC5) is highly expressed in striatal medium spiny neurons (MSNs), and is known to play an important role in mediating striatal dopaminergic signaling. Dopaminergic signaling from the D 1 expressing MSNs of the direct pathway, as well as the D 2 expressing MSNs of the indirect pathway both function through the regulation of AC5 activity, controlling the production of the 2nd messenger cAMP, and subsequently the downstream effectors. Here, we used a newly developed cell line that used Crispr-Cas9 to eliminate the predominant adenylyl cyclase isoforms to more accurately characterize a series of AC5 gain-of-function mutations which have been identified in ADCY5-related dyskinesias. Our results demonstrate that these AC5 mutants exhibit enhanced activity to Gα s -mediated stimulation in both cell and membrane-based assays. We further show that the increased cAMP response at the membrane effectively translates into increased downstream gene transcription in a neuronal model. Subsequent analysis of inhibitory pathways show that the AC5 mutants exhibit significantly reduced inhibition following D 2 dopamine receptor activation. Finally, we demonstrate that an adenylyl cyclase "P-site" inhibitor, SQ22536 may represent an effective future therapeutic mechanism by preferentially inhibiting the overactive AC5 gain-of-function mutants., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: We previously found that mirabegron exerts a relaxant effect in the presence of the β 3 -adrenoceptor antagonist SR58894A during carbachol-induced contraction in human and pig detrusor. The aim of this study was to explore the possible mechanism underlying the relaxant effects of mirabegron using detrusor smooth muscle., Methods: Human tissue was obtained from urinary bladders of patients undergoing radical cystectomy at Kyushu University and Harasanshin Hospital. Pig tissue was obtained from an abattoir. Tension force (organ bath experiments) was measured in intact or permeabilised (α-toxin or β-escin) detrusor smooth muscle strips. The contribution of cAMP-dependent signaling and the inhibition of Ca 2+ sensitization to the relaxant effects of mirabegron were characterized using 1 μM SR58894A, 100 μM SQ22536 (an adenylyl cyclase inhibitor), 10 μM H-89 (a protein kinase [PK] A inhibitor), 10 μM Y-27632 (a selective Rho kinase inhibitor), and 10 μM GF-109203X (a selective PKC inhibitor)., Results: 30 μM Mirabegron impaired carbachol (0.03-1 μM)-induced contraction in human detrusor smooth muscle. SR58894A only partially attenuated the relaxant effects of mirabegron in human and pig detrusor strips precontracted with 1 μM carbachol. In α-toxin-permeabilized detrusor strips, tension force at 1 μM [Ca 2+ ] i was decreased by mirabegron in a concentration-dependent manner. The relaxant effect of mirabegron was only slightly attenuated by H-89 and not significantly affected by SQ22536. Y-27632 potentiated the relaxation response to mirabegron, but attenuated responses to cAMP; GF-109203X had little effect. Mirabegron but not cAMP had a notable relaxant effect in the pig detrusor smooth muscle permeabilized with β-escin., Conclusions: Mirabegron-induced relaxation of pig and human detrusor smooth muscle occurs via both a β 3 -adrenoceptor/cAMP-dependent and -independent pathway., (© 2019 John Wiley & Sons Australia, Ltd.)

The application of pulsed electromagnetic fields (PEMFs) in the prevention and treatment of osteoporosis has long been an area of interest. However, the clinical application of PEMFs remains limited because of the poor understanding of the PEMF action mechanism. Here, we report that PEMFs promote bone formation by activating soluble adenylyl cyclase (sAC), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and cAMP response element-binding protein (CREB) signaling pathways. First, it was found that 50 Hz 0.6 millitesla (mT) PEMFs promoted osteogenic differentiation of rat calvarial osteoblasts (ROBs), and that PEMFs activated cAMP-PKA-CREB signaling by increasing intracellular cAMP levels, facilitating phosphorylation of PKA and CREB, and inducing nuclear translocation of phosphorylated (p)-CREB. Blocking the signaling by adenylate cyclase (AC) and PKA inhibitors both abolished the osteogenic effect of PEMFs. Second, expression of sAC isoform was found to be increased significantly by PEMF treatment. Blocking sAC using sAC-specific inhibitor KH7 dramatically inhibited the osteogenic differentiation of ROBs. Finally, the peak bone mass of growing rats was significantly increased after 2 months of PEMF treatment with 90 min/day. The serum cAMP content, p-PKA, and p-CREB as well as the sAC protein expression levels were all increased significantly in femurs of treated rats. The current study indicated that PEMFs promote bone formation in vitro and in vivo by activating sAC-cAMP-PKA-CREB signaling pathway of osteoblasts directly or indirectly., (© 2018 Wiley Periodicals, Inc.)

Objective: This study aimed to investigate the antinociceptive effects of the selective adenylyl cyclase type 1 (AC1) inhibitor ST034307 on tooth movement nociception through orofacial nociceptive behavior tests and molecular examination., Methods: We placed fixed nickel-titanium alloy closed-coil springs around the incisors of male Sprague-Dawley rats to induce tooth movement. We subsequently administered ST034307 (3 mg/kg), for 2 days, intraperitoneally, and then subjected the rats to a battery of behavioral tests (n = 10/group) to assess orofacial nociception. The changes in the expression of key molecules in the anterior cingulate cortex were measured by ELISA (n = 8/group) and Western blotting (n = 8/group)., Results: Tooth movement increased face-grooming activities and rat grimace scale scores. Tooth movement was also associated with enhanced cyclic adenosine monophosphate (cAMP) generation as well as protein kinase A (PKA) activation. Moreover, the phosphorylation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and expression of N-methyl-d-aspartate (NMDA) receptors in the anterior cingulate cortex increased during tooth movement. ST034307 significantly decreased mouth wiping and rat grimace scale scores, accompanied by reductions in cAMP generation, PKA activation, AMPA receptor phosphorylation, and NMDA receptor expression in the anterior cingulate cortex., Conclusions: These results suggest that adenylyl cyclase type 1 plays an important role in the development of orthodontic tooth movement nociception. Furthermore, ST034307 can be used as an effective pharmacotherapy for orthodontic nociception., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Manganese is a neurotoxin causing manganism, a Parkinson-like clinical disorder. Manganese has been shown to interfere with dopaminergic neurotransmission, but the neurotoxic mechanism involved is not fully resolved. In the bivalve mollusc Crassostrea virginica also known as the eastern oyster, beating rates of lateral cilia of the gill are controlled by dopaminergic-serotonergic innervation originating from their cerebral and visceral ganglia. Terminal release of dopamine activates D2-like receptors on these gill cells inhibiting adenylyl cyclase and slowing cilia beating rates. In C. virginica, manganese treatment disrupts this dopaminergic innervation of the gill, preventing the normal cilio-inhibitory response of lateral cells to dopamine. In this study an adenylyl cyclase activator (forskolin) and two different inhibitors (MDL-12,330A and SQ 22,536) were used to determine if manganese had any effects on the adenylyl cyclase step of the dopamine D2 receptor signal transduction pathway. The results showed that neither the adenylyl cyclase activator nor the inhibitors were affected by manganese in the control of lateral ciliary activity. This suggests that in C. virginica the mechanism of manganese toxicity on the dopaminergic control of lateral ciliary activity is targeting an early step in the D2R signal transduction pathway, which may involve interference with D2 receptor activation or alternatively some other downstream signaling activity that does not affect adenylyl cyclase., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Progressive movement of spermatozoa has conventionally been regarded as a good indicator of motility. However, bull spermatozoa exhibit two types of progressive movement: progressive/planar movement without rotation and progressive/helical movement with rotation. The aim of this study was to reconsider the evaluation criteria of bull ejaculated sperm motility in the context of rotation. Here, we compared the movement patterns of ejaculated spermatozoa with relatively high and low protein kinase A (PKA)-mediated signaling activities, because sperm motility is positively regulated by PKA-mediated signaling activities. We prepared sperm samples with high and low PKA-mediated signaling activities by suspending spermatozoa in media containing either the stimulator (NaHCO 3 ) or inhibitor (KH-7) of adenylyl cyclase 10, and we then investigated movement patterns and relative velocities using a microscopic high-speed camera and recording system. In the control medium without NaHCO 3 and KH-7, most spermatozoa exhibited round/planar movement without rotation and asymmetrical bends in the principal pieces. NaHCO 3 significantly promoted changes in movement patterns from round/planar movement to progressive/planar movement (without rotation) as well as symmetrization of flagellar bends and increased relative velocities. KH-7 significantly increased spermatozoa exhibiting progressive/helical movement (with rotation), decreased relative velocities, and symmetrized flagellar bends with a reduction in their size. These indicate that progressive/planar movement (without rotation) and fast movement characterize the movement patterns of bull ejaculated spermatozoa with high PKA-mediated signaling activities. A sign of reduced PKA-mediated signaling activity is not only slow movement but also helical movement (with rotation). Thus, it is beneficial to add a new parameter of "rotation" to the evaluation criteria of bull ejaculated sperm motility.

Soluble adenylate cyclase (sAC) is a non-plasma membrane-bound isoform of the adenylate cyclases signaling via the canonical second messenger, 3',5'-cyclic AMP (cAMP). sAC is involved in key physiological processes such as insulin release, sperm motility, and energy metabolism. Thus, sAC has attracted interest as a putative drug target and attempts have been made to develop selective inhibitors. Since sAC has a binding constant for its substrate, ATP, in the millimolar range, reductions in mitochondrial ATP production may be part of the mechanism-of-action of sAC inhibitors and the potential of these compounds to study the physiological outcomes of inhibition of sAC might be severely hampered by this. Here, we evaluate the effects of two commonly employed inhibitors, 2-OHE and KH7, on mitochondrial ATP production and energy metabolism. For comparison, we included a recently identified inhibitor of sAC, bithionol. Employing mitochondria isolated from mouse brain, we show that all three compounds are able to curb ATP production albeit via distinct mechanisms. Bithionol and KH7 mainly inhibit ATP production by working as a classical uncoupler whereas 2-OHE mainly works by decreasing mitochondrial respiration. These findings were corroborated by investigating energy metabolism in acute brain slices from mice. Since all three sAC inhibitors are shown to curb mitochondrial ATP production and affect energy metabolism, caution should be exercised when employed to study the physiological roles of sAC or for validating sAC as a drug target., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Introduction: Enterococcus faecalis is correlated with oral diseases including recurrent root canal treatment failure because of its biofilm formation ability and various virulence factors. Cyclic di-AMP (c-di-AMP) is an omnipresent second messenger involved in many crucial cellular physiological processes, including biofilm formation. ST056083 is a small molecule working as an inhibitor of the c-di-AMP synthetase DNA integrity scanning protein (DisA) in vitro. In this study, the impact of ST056083 on E. faecalis DisA activity, bacterial growth, and biofilm formation was tested., Methods: The binding affinity between the protein and ligand was evaluated using the Amber score, and the binding mode was analyzed and visualized using UCSF Chimera (Resource for Biocomputing, Visualization, and Informatics, University of California, San Francisco, San Francisco, CA). The effect of ST056083 on E. faecalis DisA was evaluated using the coralyne assay. The effect of ST056083 on E. faecalis biofilm formation was determined by the biofilm quantification assay, scanning electron microscopic examination, and 3-dimensional confocal laser scanning microscopic assay. The effect of ST056083 on E. faecalis exopolysaccharide synthesis was measured by the anthrone-sulfuric method., Results: We expressed and purified E. faecalis DisA in vitro and confirmed the inhibitory effect of ST056083 on its biological activity. In addition, we showed the inhibitory effect of ST056083 on E. faecalis growth, biofilm formation, and exopolysaccharide synthesis., Conclusions: Our findings enhance the understanding of the physiological role of c-di-AMP in E. faecalis and represent a preliminary study on the ST056083 inhibitory effect and mechanism., (Copyright © 2018 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Selective estrogen receptor modulators (SERMs) target estrogen receptors (ERs) to treat breast cancer and osteoporosis. Several SERMs exhibit anti-cancer activity not related to ERs. To discover novel anti-cancer drugs acting via ER-independent mechanisms, derivatives of the SERM tamoxifen, known as the "ridaifen" compounds, have been developed that exhibit reduced or no ER affinity, while maintaining cytotoxicity. Tamoxifen and other SERMs bind to cannabinoid receptors with moderate affinity. Therefore, ER-independent effects of SERMs might be mediated via cannabinoid receptors. This study determined whether RID-B, a first generation ridaifen compound, exhibits affinity and/or activity at CB 1 and/or CB 2 cannabinoid receptors. RID-B binds with high affinity (K i  = 43.7 nM) and 17-fold selectivity to CB 2 over CB 1 receptors. RID-B acts as an inverse agonist at CB 2 receptors, modulating G-protein and adenylyl cyclase activity with potency values predicted by CB 2 affinity. Characteristic of an antagonist, RID-B co-incubation produces a parallel-rightward shift in the concentration-effect curve of CB 2 agonist WIN-55,212-2 to inhibit adenylyl cyclase activity. CB 2 inverse agonists are reported to exhibit anti-inflammatory and anti-ostoeclastogenic effects. In LPS-activated macrophages, RID-B exhibits anti-inflammatory effects by reducing levels of nitric oxide (NO), IL-6 and IL-1α, but not TNFα. Only reduction of NO concentration by RID-B is mediated by cannabinoid receptors. RID-B also exhibits pronounced anti-osteoclastogenic effects, reducing the number of osteoclasts differentiating from primary bone marrow macrophages in a cannabinoid receptor-dependent manner. In summary, the tamoxifen derivative RID-B, developed with reduced affinity for ERs, is a high affinity selective CB 2 inverse agonist with anti-inflammatory and anti-osteoclastogenic properties., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Sympathetic activation causes clinically important arrhythmias including atrial fibrillation (AF) and ventricular tachyarrhythmia. Although the usefulness of β-adrenergic receptor blockade therapy is widely accepted, its multiple critical side effects often prevent its initiation or continuation. The aim of this study is to determine the advantages of vidarabine, an adenylyl cyclase (AC)-targeted anti-sympathetic agent, as an alternative treatment for arrhythmia. We found that vidarabine, which we identified as a cardiac AC inhibitor, consistently shortens AF duration and reduces the incidence of sympathetic activation-induced ventricular arrhythmias. In atrial and ventricular myocytes, vidarabine inhibits adrenergic receptor stimulation-induced RyR2 phosphorylation, sarcoplasmic reticulum (SR) Ca 2+ leakage, and spontaneous Ca 2+ release from SR, the last of which has been considered as a potential arrhythmogenic trigger. Moreover, vidarabine also inhibits sympathetic activation-induced reactive oxygen species (ROS) production in cardiac myocytes. The pivotal role of vidarabine's inhibitory effect on ROS production with regard to its anti-arrhythmic property has also been implied in animal studies. In addition, as expected, vidarabine exerts an inhibitory effect on AC function, which is more potent in the heart than elsewhere. Indexes of cardiac function including ejection fraction and heart rate were not affected by a dosage of vidarabine sufficient to exert an anti-arrhythmic effect. These findings suggest that vidarabine inhibits catecholamine-induced AF or ventricular arrhythmia without deteriorating cardiac function in mice.

Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels have important functions in controlling neuronal excitability and generating rhythmic oscillatory activity. The role of tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) in regulation of hyperpolarization-activated inward current, I h , in the thalamocortical system and its functional relevance for the physiological thalamocortical oscillations were investigated. A significant decrease in I h current density, in both thalamocortical relay (TC) and cortical pyramidal neurons was found in TRIP8b-deficient mice (TRIP8b -/- ). In addition basal cAMP levels in the brain were found to be decreased while the availability of the fast transient A-type K + current, I A , in TC neurons was increased. These changes were associated with alterations in intrinsic properties and firing patterns of TC neurons, as well as intrathalamic and thalamocortical network oscillations, revealing a significant increase in slow oscillations in the delta frequency range (0.5-4 Hz) during episodes of active-wakefulness. In addition, absence of TRIP8b suppresses the normal desynchronization response of the EEG during the switch from slow-wave sleep to wakefulness. It is concluded that TRIP8b is necessary for the modulation of physiological thalamocortical oscillations due to its direct effect on HCN channel expression in thalamus and cortex and that mechanisms related to reduced cAMP signaling may contribute to the present findings.

Enzymatic substrate selectivity is critical for the precise control of metabolic pathways. In cases where chemically related substrates are present inside cells, robust mechanisms of substrate selectivity are required. Here, we report the mechanism utilized for catalytic ATP versus GTP selectivity during adenylate kinase (Adk) -mediated phosphorylation of AMP. Using NMR spectroscopy we found that while Adk adopts a catalytically competent and closed structural state in complex with ATP, the enzyme is arrested in a catalytically inhibited and open state in complex with GTP. X-ray crystallography experiments revealed that the interaction interfaces supporting ATP and GTP recognition, in part, are mediated by coinciding residues. The mechanism provides an atomic view on how the cellular GTP pool is protected from Adk turnover, which is important because GTP has many specialized cellular functions. In further support of this mechanism, a structure-function analysis enabled by synthesis of ATP analogs suggests that a hydrogen bond between the adenine moiety and the backbone of the enzyme is vital for ATP selectivity. The importance of the hydrogen bond for substrate selectivity is likely general given the conservation of its location and orientation across the family of eukaryotic protein kinases., Competing Interests: The authors declare no conflict of interest.

Diabetes mellitus is a chronic disease in which the pancreas no longer produces enough insulin. Pancreatic alpha cell mass increases in response to insufficient insulin secretion. However, the reason for this increase is not clear. It is possible that the increased alpha-cells may stimulate compensatory insulin release in response to the insufficient insulin such as insulin resistance. In this study, we investigated whether glucagon and glucagon-like peptide-1 (GLP-1), hormones produced by alpha cells, contribute to insulin secretion in INS-1 cells, a beta cell line. We confirmed that alpha cell area in the pancreatic islets and glucagon secretion were increased in HFD-induced obese mice. Co-treatment with glucagon and exendin-4 (Ex-4), a GLP-1 receptor agonist, additively increased glucose-stimulated insulin secretion in INS-1 cells. In parallel, cAMP production was also additively increased by co-treatment with these hormones. The increase of insulin secretion by Ex-4 in the presence of high glucose was inhibited by 2'5'-dideoxyadenosine, a transmembrane adenylyl cyclase inhibitor, but not by KH-7, a soluble adenylyl cyclase inhibitor. The increase of insulin secretion by glucagon in INS-1 cells was inhibited by both 2'5'-dideoxyadenosine and KH-7. We suggest that glucagon and GLP-1 produced from alpha cells additively increase cAMP and insulin secretion in the presence of high glucose via distinct adenylyl cyclases in INS-1 cells, and this may contribute to the compensatory increase of insulin secretion by an increase of pancreatic alpha cell mass under conditions of insulin resistance., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Melanocyte development and differentiation are regulated by cAMP, which is produced by the adenylate cyclase (AC) enzyme upon activation of the melanocortin-1-receptor (MC1R). Individuals carrying single amino acid substitution variants of MC1R have impaired cAMP signaling and higher risk of melanoma. However, the contribution of AC to this risk is not clear. Downstream of AC, the phosphorylated transcription factor, cyclic AMP Responsive Element Binding Protein (pCREB), which is activated by protein kinase A, regulates the expression of several genes including the melanocyte master regulator MITF. The roles of AC and CREB in melanoma development and growth are not well understood. Here, we investigated the effect of topical application of AC inhibitor on Braf CA /Pten -/- mouse melanoma development. We show that AC inhibitor delays melanoma growth independent of MAPK pathway activity and melanin content. Next, employing a primary melanoma tissue microarray and quantitative immunohistochemistry, we show that pCREB levels are positively correlated with the proliferative status of melanoma, but low pCREB expression is associated with tumor aggressiveness and metastatic recurrence. These data suggest that low cAMP signaling inhibits tumor growth but is a predictor of melanoma aggressiveness., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Inhibition of Bordetella pertussis adenylate cyclase toxin (ACT) and Bacillus anthracis edema factor (EF), key virulence factors with adenylate cyclase activity, represents a potential method for treating or preventing toxemia related to whooping cough and anthrax, respectively. Novel α-branched acyclic nucleoside phosphonates (ANPs) having a hemiaminal ether moiety were synthesized as potential inhibitors of bacterial adenylate cyclases. ANPs prepared as bisamidates were not cytotoxic, but did not exhibit any profound activity (IC 50 >10 μm) toward ACT in J774A.1 macrophages. The apparent lack of activity of the bisamidates is speculated to be due to the inefficient formation of the biologically active species (ANPpp) in the cells. Conversely, two 5-haloanthraniloyl-substituted ANPs in the form of diphosphates were shown to be potent ACT and EF inhibitors with IC 50 values ranging from 55 to 362 nm., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive loss of dopaminergic neurons in the substantia nigra of the human brain, leading to depletion of dopamine production. Dopamine replacement therapy remains the mainstay for attenuation of PD symptoms. Nonetheless, the potential benefit of current pharmacotherapies is mostly limited by adverse side effects, such as drug-induced dyskinesia, motor fluctuations and psychosis. Non-dopaminergic receptors, such as human A2A adenosine receptors, have emerged as important therapeutic targets in potentiating therapeutic effects and reducing the unwanted side effects. In this study, new chemical entities targeting both human A2A adenosine receptor and dopamine D2 receptor were designed and evaluated. Two computational methods, namely support vector machine (SVM) models and Tanimoto similarity-based clustering analysis, were integrated for the identification of compounds containing indole-piperazine-pyrimidine (IPP) scaffold. Subsequent synthesis and testing resulted in compounds 5 and 6, which acted as human A2A adenosine receptor binders in the radioligand competition assay (Ki = 8.7-11.2 μM) as well as human dopamine D2 receptor binders in the artificial cell membrane assay (EC50 = 22.5-40.2 μM). Moreover, compound 5 showed improvement in movement and mitigation of the loss of dopaminergic neurons in Drosophila models of PD. Furthermore, in vitro toxicity studies on compounds 5 and 6 did not reveal any mutagenicity (up to 100 μM), hepatotoxicity (up to 30 μM) or cardiotoxicity (up to 30 μM).

Scope: Piperonal is an aromatic compound found in vanilla and has a floral odor resembling vanillin. This study was aimed to test whether piperonal attenuates visceral adiposity induced by a high-fat diet (HFD) in mice and to explore the underlying molecular mechanisms., Methods and Results: Male C57BL/6N mice were fed a normal diet, HFD, or 0.05% piperonal-supplemented HFD (PSD) for 10 weeks. PSD-fed mice showed attenuation of body weight gain, total visceral fat pad weights, and plasma lipid levels compared to HFD-fed mice. Piperonal supplementation of the HFD increased the mRNA expression of certain isotypes of adenylate cyclase (Adcy) and protein kinase A (PKA) in the white adipose tissue (WAT) of mice. The adipogenesis-related genes were downregulated, whereas fatty acid oxidation- and thermogenesis-related genes were upregulated in the WAT of PSD-fed mice compared to those in HFD-fed mice. Piperonal directly activated Adcy by decreasing the K m for its substrate (ATP) in plasma membranes prepared from the WAT of mice. Furthermore, piperonal-induced inhibition of adipocyte differentiation and elevation of Adcy and PKA activities in 3T3-L1 cells were abrogated by an Adcy inhibitor., Conclusion: The anti-adipogenic effect of piperonal in mice fed the high-fat diet appears to be associated with increased Adcy-PKA signaling in WAT., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cell invasion by Trypanosoma cruzi and its intracellular replication are essential for progression of the parasite life cycle and development of Chagas disease. Prostaglandin E2 (PGE 2 ) and other eicosanoids potently modulate host response and contribute to Chagas disease progression. In this study, we evaluated the effect of aspirin (ASA), a non-selective cyclooxygenase (COX) inhibitor on the T. cruzi invasion and its influence on nitric oxide and cytokine production in human monocytes. The pretreatment of monocytes with ASA or SQ 22536 (adenylate-cyclase inhibitor) induced a marked inhibition of T. cruzi infection. On the other hand, the treatment of monocytes with SQ 22536 after ASA restored the invasiveness of T. cruzi. This reestablishment was associated with a decrease in nitric oxide and PGE 2 production, and also an increase of interleukin-10 and interleukin-12 by cells pre-treated with ASA. Altogether, these results reinforce the idea that the cyclooxygenase pathway plays a fundamental role in the process of parasite invasion in an in vitro model of T. cruzi infection., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Purpose: To validate the increase in intraocular pressure (IOP) caused by soluble adenylyl cyclase (sAC) inhibitors and determine reasons behind variation in IOP measurements performed by tonometry., Methods: C57BL/6J mice were administered DMSO solubilized sAC inhibitors (KH7 or LRE-1) by intraperitoneal injection. Two hours post-treatment, mice were anesthetized with avertin or ketamine/xylazine/acepromazine (KXA). IOP was measured by a rebound tonometer or direct cannulation of the anterior chamber. Spectral-domain optical coherence tomography was used to measure anterior chamber depth and corneal thickness in live mice. Outflow facility was measured in perfused, enucleated mouse eyes., Results: Compared with DMSO controls, KH7 treatment caused an increased IOP in avertin- and KXA-anesthetized mice when measured by direct cannulation [avertin: 14.4 ± 2.1 mmHg vs. 11.1 ± 1.0 mmHg (P = 0.003); KXA: 14.4 ± 1.0 mmHg vs. 11.3 ± 0.8 mmHg (P < 0.001)] and tonometry [avertin: 10.8 ± 1.4 mmHg vs. 7.4 ± 0.6 mmHg (P < 0.001); KXA: 11.9 ± 0.9 mmHg vs. 10.3 ± 1.7 mmHg (P = 0.283)]. However, treatment with KH7 in nonanesthetized mice showed a significant decrease in IOP measured by tonometry and compared with DMSO-treated animals [13.1 ± 2.6 mmHg vs. 15.6 ± 0.5 mmHg (P = 0.003)]. Both KH7- and DMSO-treated groups anesthetized with avertin showed increased corneal thickness, whereas KH7-treated mice anesthetized with KXA exhibited a shallower anterior chamber compared with untreated mice. KH7 decreased outflow facility by 85.1% in nonanesthetized, enucleated eyes (P < 0.003)., Conclusions: Systemically administered DMSO and anesthesia have significant effects on anterior chamber characteristics, resulting in altered IOP readings measured by tonometry. In the presence of DMSO and anesthesia, tonometry IOP readings should be confirmed with direct cannulation.

Phototactic behaviours are observed from prokaryotes to amphibians and are a basic form of orientation. We showed that the marbled crayfish displays phototaxis in which the behavioural response reversed from negative to positive depending on external light conditions. Animals reared in a 12-L/12-D light cycle showed negative phototaxis during daytime and positive phototaxis during night-time. Animals reared under constant light conditioning showed negative phototaxis during day- and night-time, while animals reared under constant dark conditioning showed positive phototaxis during day- and night-time. Injection of serotonin leads to a reversal of negative to positive phototaxis in both light/dark-reared and light/light-reared animals while injection of dopamine induced reversed negative phototaxis in dark/dark-reared animals. Four hours of dark adaptation were enough for light/dark-reared animals to reverse phototaxis from negative to positive. Injection of a serotonin 5HT 1 receptor antagonist blocked the reverse phototaxis while serotonin 5HT 2 receptor antagonists had no effects. Similarly, dark/dark-reared animals reversed to showing negative phototaxis after 4 h of light adaptation. Injection of a dopamine DA 1 receptor antagonist blocked this reverse phototaxis, while dopamine DA 2 receptor antagonists had no effects. Injection of a cAMP analogue into light/dark-reared animals blocked reverse phototaxis after dark adaptation, while adenylate cyclase inhibitor in dark/dark-reared animals blocked reverse phototaxis after light adaptation. These results strongly suggest that serotonin mediates positive phototaxis owing to decreased cAMP levels, while dopamine-mediated negative phototaxis occurs due to increased cAMP levels. Supporting this, the ratio of serotonin to dopamine in the brain was much higher in dark/dark-reared than light/dark-reared animals., (© 2017 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Background: Based on the traditional application of traditional Chinese Medicines (TCMs), Ephedra Herba (EH) is used to cure cold fever by inducing sweating, whereas Ephedra Radix (ER) is used to treat hyperhidrosis. Although they come from the same plant, Ephedra sinica Stapf, but have play opposing roles in clinical applications. EH is known to contain ephedrine alkaloids, which is the driver of the physiological changes in sweating, heart rate and blood pressure. However, the active pharmacological ingredients (APIs) of ER and the mechanisms by which it restricts sweating remain unknown., Purpose: The current work aims to discover the hidroschesis APIs from ER, as well as to establish its action mechanism., Methods: UPLC-Q/TOF-MS, PCA, and heat map were utilized for identifying the differences between EH and ER. HPLC integrated with a β 2 -adrenoceptor (β 2 -AR) activity luciferase reporter assay system was used to screen active inhibitors; molecular docking and a series of biological assays centered on β 2 -AR-related signaling pathways were evaluated to understand the roles of APIs., Results: The opposite effect on sweating of EH and ER can be attributed to the APIs of amphetamine-type alkaloids and flavonoid derivatives. Mahuannin B is an effective anti-hydrotic agent, inhibiting the production of cAMP via suppression of adenylate cyclase (AC) activity., Conclusion: The effects of EH and ER on sweat and β 2 -AR-related signaling pathway are opposite due to different alkaloids and flavonoids of APIs in EH and ER. The present work not only sheds light on the hidroschesis action of mahuannin B, but also presents a potential target of AC in the treatment of hyperhidrosis., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Adenylyl cyclases (ACs) generate the second messenger cAMP from ATP. Mammalian cells express nine transmembrane AC (mAC) isoforms (AC1-9) and a soluble AC (sAC, also referred to as AC10). This review will largely focus on mACs. mACs are activated by the G-protein G α s and regulated by multiple mechanisms. mACs are differentially expressed in tissues and regulate numerous and diverse cell functions. mACs localize in distinct membrane compartments and form signaling complexes. sAC is activated by bicarbonate with physiologic roles first described in testis. Crystal structures of the catalytic core of a hybrid mAC and sAC are available. These structures provide detailed insights into the catalytic mechanism and constitute the basis for the development of isoform-selective activators and inhibitors. Although potent competitive and noncompetitive mAC inhibitors are available, it is challenging to obtain compounds with high isoform selectivity due to the conservation of the catalytic core. Accordingly, caution must be exerted with the interpretation of intact-cell studies. The development of isoform-selective activators, the plant diterpene forskolin being the starting compound, has been equally challenging. There is no known endogenous ligand for the forskolin binding site. Recently, development of selective sAC inhibitors was reported. An emerging field is the association of AC gene polymorphisms with human diseases. For example, mutations in the AC5 gene ( ADCY5 ) cause hyperkinetic extrapyramidal motor disorders. Overall, in contrast to the guanylyl cyclase field, our understanding of the (patho)physiology of AC isoforms and the development of clinically useful drugs targeting ACs is still in its infancy., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

Background & Aims: Genetic defects in polycystin-1 or -2 (PC1 or PC2) cause polycystic liver disease associated with autosomal dominant polycystic kidney disease (PLD-ADPKD). Progressive cyst growth is sustained by a cAMP-dependent Ras/ERK/HIFα pathway, leading to increased vascular endothelial growth factor A (VEGF-A) signaling. In PC2-defective cholangiocytes, cAMP production in response to [Ca 2+ ] ER depletion is increased, while store-operated Ca 2+ entry (SOCE), intracellular and endoplasmic reticulum [Ca 2+ ] ER levels are reduced. We investigated whether the adenylyl cyclases, AC5 and AC6, which can be inhibited by Ca 2+ , are activated by the ER chaperone STIM1. This would result in cAMP/PKA-dependent Ras/ERK/HIFα pathway activation in PC2-defective cells, in response to [Ca 2+ ] ER depletion., Methods: PC2/AC6 double conditional knockout (KO) mice were generated (Pkd2/AC6 KO) and compared to Pkd2 KO mice. The AC5 inhibitor SQ22,536 or AC5 siRNA were used in isolated cholangiocytes while the inhibitor was used in biliary organoid and animals; liver tissues were harvested for histochemical analysis., Results: When comparing Pkd2/AC6 KO to Pkd2 KO mice, no decrease in liver cyst size was found, and cellular cAMP after [Ca 2+ ] ER depletion only decreased by 12%. Conversely, in PC2-defective cells, inhibition of AC5 significantly reduced cAMP production, pERK1/2 expression and VEGF-A secretion. AC5 inhibitors significantly reduced growth of biliary organoids derived from Pkd2 KO and Pkd2/AC6 KO mice. In vivo treatment with SQ22,536 significantly reduced liver cystic area and cell proliferation in PC2-defective mice. After [Ca 2+ ] ER depletion in PC2-defective cells, STIM1 interacts with AC5 but not with Orai1, the Ca 2+ channel that mediates SOCE., Conclusion: [Ca 2+ ] ER depletion in PC2-defective cells activates AC5 and results in stimulation of cAMP/ERK1-2 signaling, VEGF production and cyst growth. This mechanism may represent a novel therapeutic target., Lay Summary: Polycystic liver diseases are characterized by progressive cyst growth until their complications mandate surgery or liver transplantation. In this manuscript, we demonstrate that inhibiting cell proliferation, which is induced by increased levels of cAMP, may represent a novel therapeutic target to slow the progression of the disease., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Adenylyl cyclase 1 (AC1) belongs to a group of adenylyl cyclases (ACs) that are stimulated by calcium in a calmodulin-dependent manner. Studies with AC1 knockout mice suggest that inhibitors of AC1 may be useful for treating pain and opioid dependence. However, nonselective inhibition of AC isoforms could result in substantial adverse effects. We used chemical library screening to identify a selective AC1 inhibitor with a chromone core structure that may represent a new analgesic agent. After demonstrating that the compound (ST034307) inhibited Ca 2+ -stimulated adenosine 3',5'-monophosphate (cAMP) accumulation in human embryonic kidney (HEK) cells stably transfected with AC1 (HEK-AC1 cells), we confirmed selectivity for AC1 by testing against all isoforms of membrane-bound ACs. ST034307 also inhibited AC1 activity stimulated by forskolin- and Gα s -coupled receptors in HEK-AC1 cells and showed inhibitory activity in multiple AC1-containing membrane preparations and mouse hippocampal homogenates. ST034307 enhanced μ-opioid receptor (MOR)-mediated inhibition of AC1 in short-term inhibition assays in HEK-AC1 cells stably transfected with MOR; however, the compound blocked heterologous sensitization of AC1 caused by chronic MOR activation in these cells. ST034307 reduced pain responses in a mouse model of inflammatory pain. Our data indicate that ST034307 is a selective small-molecule inhibitor of AC1 and suggest that selective AC1 inhibitors may be useful for managing pain., (Copyright © 2017, American Association for the Advancement of Science.)

Fragile X syndrome (FXS), caused by the loss of functional FMRP, is a leading cause of autism. Neurons lacking FMRP show aberrant mRNA translation and intracellular signalling. Here, we identify that, in Fmr1 knockout neurons, type 1 adenylyl cyclase (Adcy1) mRNA translation is enhanced, leading to excessive production of ADCY1 protein and insensitivity to neuronal stimulation. Genetic reduction of Adcy1 normalizes the aberrant ERK1/2- and PI3K-mediated signalling, attenuates excessive protein synthesis and corrects dendritic spine abnormality in Fmr1 knockout mice. Genetic reduction of Adcy1 also ameliorates autism-related symptoms including repetitive behaviour, defective social interaction and audiogenic seizures. Moreover, peripheral administration of NB001, an experimental compound that preferentially suppresses ADCY1 activity over other ADCY subtypes, attenuates the behavioural abnormalities in Fmr1 knockout mice. These results demonstrate a connection between the elevated Adcy1 translation and abnormal ERK1/2 signalling and behavioural symptoms in FXS.

Baicalin has been reported to attenuate lung edema in the process of lung injury. However, the effect of baicalin on alveolar fluid clearance (AFC) and epithelial sodium channel (ENaC) expression has not been tested. Sprague-Dawley rats were anesthetized and intratracheally injected with either 1 mg/kg lipopolysaccharide (LPS) or saline vehicle. Baicalin with various concentrations (10, 50, and 100 mg/kg) was injected intraperitoneally 30 min before administration of LPS. Then lungs were isolated for measurement of AFC, cyclic adenosine monophosphate (cAMP) level, and cellular localization of α-ENaC. Moreover, mouse alveolar type II (ATII) epithelial cell line was incubated with baicalin (30 μmol/L), adenylate cyclase inhibitor SQ22536 (10 μmol/L), or cAMP-dependent protein kinase inhibitor (PKA) KT5720 (0.3 μmol/L) 15 min before LPS (1 μg/mL) incubation. Protein expression of α-ENaC was detected by Western blot. Baicalin increased cAMP concentration and AFC in a dose-dependent manner in rats with LPS-induced acute lung injury. The increase of AFC induced by baicalin was associated with an increase in the abundance of α-ENaC protein. SQ22536 and KT5720 prevented the increase of α-ENaC expression caused by baicalin in vitro. These findings suggest that baicalin prevents LPS-induced reduction of AFC by upregulating α-ENaC protein expression, which is activated by stimulating cAMP/PKA signaling pathway.

Cannabinoid receptors, like other GPCRs, signal via a spectrum of related signaling pathways. Recently, monitoring GPCR-mediated cAMP signaling has become significantly easier with the development of genetically encoded, transfectable cAMP biosensors. Cell lines transfected with these biosensors can be monitored continuously, allowing the analysis of receptor-mediated signaling in unprecedented detail. Here, we describe a protocol for transfectable biosensors which report cellular cAMP concentrations by bioluminescence resonance energy transfer (BRET). This assay system has been utilized to elucidate the temporal nature of agonists and allosteric modulators of the cannabinoid receptor CB1. In particular, the CB1 allosteric modulator ORG27569 has been shown to modify receptor agonism in a time-dependent fashion; a characteristic which would not have been observed via traditional endpoint methods of detecting cAMP signaling. BRET cAMP biosensors are suitable for miniaturization and automation, and as such are valuable and cost-effective tools for moderate- to high-throughput experimental protocols., (© 2017 Elsevier Inc. All rights reserved.)

Bordetella pertussis adenylate cyclase toxin (ACT) and Bacillus anthracis edema factor (EF) are key virulence factors with adenylate cyclase (AC) activity that substantially contribute to the pathogenesis of whooping cough and anthrax, respectively. There is an urgent need to develop potent and selective inhibitors of bacterial ACs with prospects for the development of potential antibacterial therapeutics and to study their molecular interactions with the target enzymes. Novel fluorescent 5-chloroanthraniloyl-substituted acyclic nucleoside phosphonates (Cl-ANT-ANPs) were designed and synthesized in the form of their diphosphates (Cl-ANT-ANPpp) as competitive ACT and EF inhibitors with sub-micromolar potency (IC 50 values: 11-622 nm). Fluorescence experiments indicated that Cl-ANT-ANPpp analogues bind to the ACT active site, and docking studies suggested that the Cl-ANT group interacts with Phe306 and Leu60. Interestingly, the increase in direct fluorescence with Cl-ANT-ANPpp having an ester linker was strictly calmodulin (CaM)-dependent, whereas Cl-ANT-ANPpp analogues with an amide linker, upon binding to ACT, increased the fluorescence even in the absence of CaM. Such a dependence of binding on structural modification could be exploited in the future design of potent inhibitors of bacterial ACs. Furthermore, one Cl-ANT-ANP in the form of a bisamidate prodrug was able to inhibit B. pertussis ACT activity in macrophage cells with IC 50 =12 μm., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: We previously showed that the levels of both Giα-2 and Giα-3 proteins were augmented in spontaneously hypertensive rats (SHRs) before the onset of hypertension. In addition, intraperitoneal injection of pertussis toxin, which inactivates both Giα proteins, prevented the development of hypertension in SHRs. The aim of the present study was to determine the specific contributions of Giα-2 and Giα-3 proteins to the development of hypertension., Methods and Results: Antisense oligodeoxynucleotide of Giα-2 and Giα-3 encapsulated in PEG/DOTAP/DOPE cationic liposomes were administrated intravenously into 3-week-old prehypertensive SHRs and Wistar Kyoto rats, whereas the control Wistar Kyoto rats and SHRs received PBS, empty liposomes, or sense. The knockdown of Giα-2 but not Giα-3 protein attenuated tachycardia and prevented the development of hypertension up to age 6 weeks; thereafter, blood pressure started increasing and reached the same level as that of untreated SHRs at 9 weeks. Furthermore, Giα-2 and Giα-3 antisense oligodeoxynucleotide treatments significantly decreased the enhanced levels of Giα-2 and Giα-3 proteins, respectively, and enhanced levels of superoxide anion and NADPH oxidase activity in heart, aorta, and kidney and hyperproliferation of vascular smooth muscle cells from SHRs aged 6 weeks. In addition, antisense oligodeoxynucleotide treatment with Giα-2 but not Giα-3 restored enhanced inhibition of adenylyl cyclase by oxotremorine to WKY levels., Conclusions: These results suggested that the enhanced expression of Giα-2 but not Giα-3 protein plays an important role in the pathogenesis of hypertension and tachycardia in SHRs., (© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

The prototypical second messenger cAMP regulates a wide variety of physiological processes. It can simultaneously mediate diverse functions by acting locally in independently regulated microdomains. In mammalian cells, two types of adenylyl cyclase generate cAMP: G-protein-regulated transmembrane adenylyl cyclases and bicarbonate-, calcium- and ATP-regulated soluble adenylyl cyclase (sAC). Because each type of cyclase regulates distinct microdomains, methods to distinguish between them are needed to understand cAMP signaling. We developed a mass-spectrometry-based adenylyl cyclase assay, which we used to identify a new sAC-specific inhibitor, LRE1. LRE1 bound to the bicarbonate activator binding site and inhibited sAC via a unique allosteric mechanism. LRE1 prevented sAC-dependent processes in cellular and physiological systems, and it will facilitate exploration of the therapeutic potential of sAC inhibition., Competing Interests: Drs. Buck, Levin and Zippin own equity interest in CEP Biotech which has licensed commercialization of a panel of monoclonal antibodies directed against sAC. All other authors declare that they have no conflicts of interest with the contents of this article.