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Neurally mediated syncope (NMS) is associated with a sudden loss of consciousness. However, the diagnostic tools and measures for prognosis management are limited. To overcome these limitations, the differences in the binding energies of Giα-protein-coupled receptors to the Glu9 and Glu12 residues on the α2B-AR gene were elucidated through the analysis of α2B-AR gene polymorphism. The suppression of the activity of adenylate cyclase (AC), which is involved in vasoconstriction, may be related to the onset of NMS. The head-up tilt (HUT) test results indicated differences in systolic blood pressure (SBP) and AC activity between patients with vasodepressor (VT)-NMS and healthy volunteers. Patients with VT-NMS had increased AC activity and decreased SBP. Conversely, in healthy volunteers, no changes in AC activity or SBP were found. These findings suggest that a high SBP and elevated AC activity at rest are likely to cause syncope. A high incidence of cardiovascular events is found in patients with negative HUT test results, highlighting the importance of investigating the cause of syncope in cases where the HUT test results are negative. Overall, our results may provide a means of assessing the risk of NMS development within healthy populations and underscore the importance of subsequent treatments for NMS. [ABSTRACT FROM AUTHOR]

Adenylyl cyclase (AC) is an enzyme that catalyses the formation of the second messenger molecule, 3′,5′-cyclic adenosine monophosphate (cAMP) from 5′-adenosine triphosphate (ATP). cAMP, in turn, regulates key physiological processes such as cell division, growth, reproduction, development and response to stress. However, while cAMP is increasingly becoming an important signalling molecule in higher plants, the identification of plant ACs has somewhat remained so slow. In Arabidopsis thaliana alone, only twelve ACs have so far been identified, yet considering the number and diverse nature of processes known to be cAMP-dependent in this plant, these identified ACs are still very much few to account for that. Notably, an additional protein in this plant, termed linker histone-like (AtLHL) protein (encoded by the At3g18035 gene), is annotated to be an AC as result of it containing a putative centre identical to the one commonly found in the other twelve previously confirmed Arabidopsis ACs. In addition, AtLHL is mostly involved in a number of key cellular processes such as heterochromatin formation, DNA repair, apoptosis, embryogenesis, reproduction and disease resistance that are all modulated by cAMP, yet AtLHL still remains unconfirmed as an AC. As a result, we targeted this protein in this study to determine if it is indeed an AC. To begin with, we used computational analysis to assess the 3-dimensional (3D) structure of AtLHL and found that its AC centre is solvent-exposed, amenable to the unhindered access of ATP as a substrate for catalysis. Next, we cloned, partially expressed and affinity purified a truncated version of this protein (AtLHL301−480), followed by assessment of its probable AC activity. Through enzyme immunoassay and mass spectrometry, we showed that the recombinant AtLHL301−480 protein can generate cAMP from ATP in vitro in a manganese-dependent manner that is enhanced by calcium and hydrogen carbonate. In addition, we also showed that the recombinant AtLHL301−480 protein can complement AC-deficiency (cyaA mutation) in SP850 cells when expressed in this mutant Escherichia coli host strain. We then used electrochemistry to evaluate the molecular interaction of AtLHL301−480 with its co-factors and modulators during catalysis and activation, respectively, and found that the protein does this physically. This observation then prompted us to specifically search for the presence (and possibly frequency) of calcium-binding sites within the AtLHL protein. Through in silico analysis and bioinformatic studies, a single binding site in form of a 16-residue calmodulin-binding sequence was predicted. Lastly, we then evaluated the reaction kinetics of AtLHL301−480 and determined that the protein has a Km constant of 0.7 mM and a Vmax constant of 9.2 fmol/min/μg protein. All in all, our study provided adequate evidence in a multi-faceted manner that LHL from A. thaliana is a bona fide AC, whose activity might be involved in control and molecular regulation of the various functions of this protein in this plant. [ABSTRACT FROM AUTHOR]

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

Obesity and major depressive disorder (MDD) are both significant health issues that have been increasing in prevalence and are associated with multiple comorbidities. Obesity and MDD have been shown to be bidirectionally associated, and they are both influenced by genetics and environmental factors. However, the molecular mechanisms that link these two diseases are not yet fully understood. It is possible that these diseases are connected through the actions of the cAMP/protein kinase A (PKA) pathway. Within this pathway, adenylate cyclase 3 (Adcy3) has emerged as a key player in both obesity and MDD. Numerous genetic variants in Adcy3 have been identified in humans in association with obesity. Rodent knockout studies have also validated the importance of this gene for energy homeostasis. Furthermore, Adcy3 has been identified as a top candidate gene and even a potential blood biomarker for MDD. Adcy3 and the cAMP/PKA pathway may therefore serve as an important genetic and functional link between these two diseases. In this mini-review, we discuss the role of both Adcy3 and the cAMP/PKA pathway, including specific genetic mutations, in both diseases. Understanding the role that Adcy3 mutations play in obesity and MDD could open the door for precision medicine approaches and treatments for both diseases that target this gene. [ABSTRACT FROM AUTHOR]

ABSTRACTBordetella species that cause respiratory infections in mammals include B. pertussis, which causes human whooping cough, and B. bronchiseptica, which infects nearly all mammals. Both bacterial species produce filamentous hemagglutinin (FhaB) and adenylate cyclase toxin (ACT), prominent surface-associated and secreted virulence factors that contribute to persistence in the lower respiratory tract by inhibiting clearance by phagocytic cells. FhaB and ACT proteins interact with themselves, each other, and host cells. Using immunoblot analyses, we showed that ACT binds to FhaB on the bacterial surface before it can be detected in culture supernatants. We determined that SphB1, a surface protease identified based on its requirement for FhaB cleavage, is also required for ACT cleavage, and we determined that the presence of ACT blocks SphB1-dependent and -independent cleavage of FhaB, but the presence of FhaB does not affect SphB1-dependent cleavage of ACT. The primary SphB1-dependent cleavage site on ACT is proximal to ACT’s active site, in a region that is critical for ACT activity. We also determined that FhaB-bound ACT on the bacterial surface can intoxicate host cells producing CR3, the receptor for ACT. In addition to increasing our understanding of FhaB, ACT, and FhaB-ACT interactions on the Bordetella surface, our data are consistent with a model in which FhaB functions as a novel toxin delivery system by binding to ACT and allowing its release upon binding of ACT to its receptor, CR3, on phagocytic cells.IMPORTANCEBacteria need to control the variety, abundance, and conformation of proteins on their surface to survive. Members of the Gram-negative bacterial genus Bordetella include B. pertussis, which causes whooping cough in humans, and B. bronchiseptica, which causes respiratory infections in a broad range of mammals. These species produce two prominent virulence factors, the two-partner secretion (TPS) effector FhaB and adenylate cyclase toxin (ACT), that interact with themselves, each other, and host cells. Here, we determined that ACT binds FhaB on the bacterial surface before being detected in culture supernatants and that ACT bound to FhaB can be delivered to eukaryotic cells. Our data are consistent with a model in which FhaB delivers ACT specifically to phagocytic cells. This is the first report of a TPS system facilitating the delivery of a separate polypeptide toxin to target cells and expands our understanding of how TPS systems contribute to bacterial pathogenesis.

ABSTRACT Noelia Lander works on cell signaling in American trypanosomes and studies the role of cyclic adenosine monophosphate (cAMP) microdomains in environmental sensing and differentiation. In this mSphere of Influence, Dr. Lander reflects on three research articles in different eukaryotic models that had impacted on the way she thinks about the regulation of cAMP signals in Trypanosoma cruzi, the etiologic agent of Chagas disease. The articles “FRET biosensor uncovers cAMP nano-domains at β-adrenergic targets that dictate precise tuning of cardiac contractility” (N. C. Surdo, M. Berrera, A. Koschinski, M. Brescia, et al., Nat Commun 8:15031, 2017, https://doi.org/10.1038/ncomms15031), “Cyclic AMP signaling and glucose metabolism mediate pH taxis by African trypanosomes” (S. Shaw, S. Knüsel, D. Abbühl, A. Naguleswaran, et al., Nat Commun 13:603, 2022, https://doi.org/10.1038/s41467-022-28293-w), and “Encystation stimuli sensing is mediated by adenylate cyclase AC2-dependent cAMP signaling in Giardia” (H. W. Shih, G. C. M. Alas, and A. R. Paredez, Nat Commun 14:7245, 2023, https://doi.org/10.1038/s41467-023-43028-1) influenced her current hypothesis that cAMP signals are generated in response to environmental cues leading to changes in membrane fluidity at the flagellar tip and the contractile vacuole complex of T. cruzi, structures where cAMP mediates key cellular processes for developmental progression.

B. parapertussis is one of the etiological agents of whooping cough. Once inhaled, the bacteria bind to the respiratory epithelium and start the infection. Little is known about this first step of host colonization and the role of the human airway epithelial barrier on B. parapertussis infection. We here investigated the outcome of the interaction of B. parapertussis with a polarized monolayer of respiratory epithelial cells. Our results show that B. parapertussis preferentially attaches to the intercellular boundaries, and causes the disruption of the tight junction integrity through the action of adenylate cyclase toxin (CyaA). We further found evidence indicating that this disruption enables the bacterial access to components of the basolateral membrane of epithelial cells to which B. parapertussis efficiently attaches and gains access to the intracellular location, where it can survive and eventually spread back into the extracellular environment. Altogether, these results suggest that the adenylate cyclase toxin enables B. parapertussis to overcome the epithelial barrier and eventually establish a niche of persistence within the respiratory epithelial cells. [ABSTRACT FROM AUTHOR]

In this case study, we report the case of a 13-year-old girl with citrullinemia type 1 (MIM #215700), an autosomal recessive inherited disorder of the urea cycle, which was confirmed by the identification of a homozygous pathogenic variant in the argininosuccinate synthetase 1 (ASS1) gene. However, the patient presented abnormal hyperkinetic movements with global developmental delay and clinical signs that were not fully consistent with those of citrullinemia type 1 or with those of her siblings with isolated citrullinemia type 1. Exome sequencing showed the presence of a de novo heterozygous pathogenic variant in the adenylate cyclase type 5 (ADCY5) gene. The variant confirmed the overlap with the so-called ADCY5-related dyskinesia with orofacial involvement, which is autosomal dominant (MIM #606703), a disorder disrupting the enzymatic conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP). In addition to the citrullinemia-related low-protein diet and arginine supplementation, the identification of this second disease led to the introduction of a treatment with caeine, which considerably improved the dyskinesia neurological picture. In conclusion, this case highlights the importance of clinical-biological confrontation for the interpretation of genetic variants, as one hereditary metabolic disease may hide another with therapeutic consequences. Summary: This article reports the misleading superposition of two inherited metabolic diseases, showing the importance of clinical-biological confrontation in the interpretation of genetic variants. [ABSTRACT FROM AUTHOR]

Migraine ranks among the most prevalent disorders worldwide, leading to disability and decreased quality of life in patients. Recently, neurogenic inflammation has been recognized as a potential underlying pathology contributing to the migraine pain pathway. Mast cells reside in the meninges and have been implicated in contributing to the pathophysiology of migraine. Here we report for the first time that the mouse Mas-Related G-protein-coupled Receptor B2 (MrgprB2), is expressed on meningeal connective tissue mast cells and contributes to Pituitary Adenylate Cyclase Activating Peptide (PACAP)-induced migraine-like pain behavior. We also found that PACAP was able to dose-dependently lead to enzyme release from human mast cells via activation of MRGPRX2; the human homolog of MrgprB2. Using a transgenic MRGPRX2 mouse, we observed significant increases in PACAP-induced migraine-like pain behavior in MRGPRX2+ mice vs mice lacking the receptor. These results reveal both MrgprB2 and MRGPRX2 as important contributors to neuropeptide-induced migraine pain. [ABSTRACT FROM AUTHOR]

Changes in cholesterol content of neuronal membranes occur during development and brain aging. Little is known about whether synaptic activity regulates cholesterol levels in neuronal membranes and whether these changes affect neuronal development and function. We generated transgenic flies that express the cholesterol-binding D4H domain of perfringolysin O toxin and found increased levels of cholesterol in presynaptic terminals of Drosophila larval neuromuscular junctions following increased synaptic activity. Reduced cholesterol impaired synaptic growth and largely prevented activity-dependent synaptic growth. Presynaptic knockdown of adenylyl cyclase phenocopied the impaired synaptic growth caused by reducing cholesterol. Furthermore, the effects of knocking down adenylyl cyclase and reducing cholesterol were not additive, suggesting that they function in the same pathway. Increasing cAMP levels using a dunce mutant with reduced phosphodiesterase activity failed to rescue this impaired synaptic growth, suggesting that cholesterol functions downstream of cAMP. We used a protein kinase A (PKA) sensor to show that reducing cholesterol levels reduced presynaptic PKA activity. Collectively, our results demonstrate that enhanced synaptic activity increased cholesterol levels in presynaptic terminals and that these changes likely activate the cAMP-PKA pathway during activity-dependent growth. [ABSTRACT FROM AUTHOR]

Protozoan parasites use cAMP signaling to precisely regulate the place and time of developmental differentiation, yet it is unclear how this signaling is initiated. Encystation of the intestinal parasite Giardia lamblia can be activated by multiple stimuli, which we hypothesize result in a common physiological change. We demonstrate that bile alters plasma membrane fluidity by reducing cholesterol-rich lipid microdomains, while alkaline pH enhances bile function. Through depletion of the cAMP producing enzyme Adenylate Cyclase 2 (AC2) and the use of a newly developed Giardia-specific cAMP sensor, we show that AC2 is necessary for encystation stimuli-induced cAMP upregulation and activation of downstream signaling. Conversely, over expression of AC2 or exogenous cAMP were sufficient to initiate encystation. Our findings indicate that encystation stimuli induce membrane reorganization, trigger AC2-dependent cAMP upregulation, and initiate encystation-specific gene expression, thereby advancing our understanding of a critical stage in the life cycle of a globally important parasite. Giardia lamblia is an established model for studying encystation. Shih et al show bile and alkaline pH induce changes in membrane ordering, upregulate cAMP, and initiate encystation gene expression. [ABSTRACT FROM AUTHOR]

Flagellar motility in sperm is activated and regulated by factors related to the eggs at fertilization. In the ascidian Ciona intestinalis, a sulfated steroid called the SAAF (sperm activating and attracting factor) induces both sperm motility activation and chemotaxis. Cyclic AMP (cAMP) is one of the most important intracellular factors in the sperm signaling pathway. Adenylyl cyclase (AC) is the key enzyme that synthesizes cAMP at the onset of the signaling pathway in all cellular functions. We previously reported that both transmembrane AC (tmAC) and soluble AC (sAC) play important roles in sperm motility in Ciona. The tmAC plays a major role in the SAAF-induced activation of sperm motility. On the other hand, sAC is involved in the regulation of flagellar beat frequency and the Ca2+-dependent chemotactic movement of sperm. In this study, we focused on the role of sAC in the regulation of flagellar motility in Ciona sperm chemotaxis. The immunochemical analysis revealed that several isoforms of sAC protein were expressed in Ciona sperm, as reported in mammals and sea urchins. We demonstrated that sAC inhibition caused strong and transient asymmetrization during the chemotactic turn, and then sperm failed to turn toward the SAAF. In addition, real-time Ca2+ imaging in sperm flagella revealed that sAC inhibition induced an excessive and prolonged Ca2+ influx to flagella. These results indicate that sAC plays a key role in sperm chemotaxis by regulating the clearance of [Ca2+]i and by modulating Ca2+-dependent flagellar waveform conversion. [ABSTRACT FROM AUTHOR]

Caveolae are tiny invaginations in the sarcolemma that buffer extra membrane and contribute to mechanical regulation of cellular function. While the role of caveolae in membrane mechanosensation has been studied predominantly in non-cardiomyocyte cells, caveolae contribution to cardiac mechanotransduction remains elusive. Here, we studied the role of caveolae in the regulation of Ca2+ signaling in atrial cardiomyocytes. In Langendorff-perfused mouse hearts, atrial pressure/volume overload stretched atrial myocytes and decreased caveolae density. In isolated cells, caveolae were disrupted through hypotonic challenge that induced a temporal (<10 min) augmentation of Ca2+ transients and caused a rise in Ca2+ spark activity. Similar changes in Ca2+ signaling were observed after chemical (methyl-β-cyclodextrin) and genetic ablation of caveolae in cardiac-specific conditional caveolin-3 knock-out mice. Acute disruption of caveolae, both mechanical and chemical, led to the elevation of cAMP level in the cell interior, and cAMP-mediated augmentation of protein kinase A (PKA)-phosphorylated ryanodine receptors (at Ser2030 and Ser2808). Caveolae-mediated stimulatory effects on Ca2+ signaling were abolished via inhibition of cAMP production by adenyl cyclase antagonists MDL12330 and SQ22536, or reduction of PKA activity by H-89. A compartmentalized mathematical model of mouse atrial myocytes linked the observed changes to a microdomain-specific decrease in phosphodiesterase activity, which disrupted cAMP signaling and augmented PKA activity. Our findings add a new dimension to cardiac mechanobiology and highlight caveolae-associated cAMP/PKA-mediated phosphorylation of Ca2+ handling proteins as a novel component of mechano-chemical feedback in atrial myocytes. [Display omitted] • Caveolae are an important but poorly understood component of membrane mechanosensing complex in cardiomyocytes. • Stretching of atrial myocytes flattens caveolae and increases membrane tension. • Caveolae disruption results in augmented Ca2+ signaling via cAMP-mediated increase in PKA-phosphorylated ryanodine receptors. • Computational modeling linked observed changes in Ca2+ signaling to a loss of caveolae-confined control of phosphodiesterases. • Caveolae-associated changes in Ca2+ signaling is a novel part of mechano-chemical feedback in atrial myocytes. [ABSTRACT FROM AUTHOR]

Photoactivated adenylate cyclases (PACs) are multidomain BLUF proteins that regulate the cellular levels of cAMP in a light-dependent manner. The signaling route and dynamics of PAC from Oscillatoria acuminata (OaPAC), which consists of a light sensor BLUF domain, an adenylate cyclase domain, and a connector helix (α3-helix), were studied by detecting conformational changes in the protein moiety. Although circular dichroism and small-angle X-ray scattering measurements did not show significant changes upon light illumination, the transient grating method successfully detected light-induced changes in the diffusion coefficient (diffusion-sensitive conformational change (DSCC)) of full-length OaPAC and the BLUF domain with the α3-helix. DSCC of full-length OaPAC was observed only when both protomers in a dimer were photoconverted. This light intensity dependence suggests that OaPAC is a cyclase with a nonlinear light intensity response. The enzymatic activity indeed nonlinearly depends on light intensity, that is, OaPAC is activated under strong light conditions. It was also found that both DSCC and enzymatic activity were suppressed by a mutation in the W90 residue, indicating the importance of the highly conserved Trp in many BLUF domains for the function. Based on these findings, a reaction scheme was proposed together with the reaction dynamics. [ABSTRACT FROM AUTHOR]

The C-C motif chemokine ligand 2 (CCL2) has been implicated in chronic pain, but its exact mechanism of peripheral sensitization is unknown. In this study, we aimed to clarify the mechanism of CCL2 regulation of ion channels. Our behavioral experiments revealed that ZD7288, a blocker of Ih current, can inhibit CFA and CCL2-mediated mechanical and thermal nociceptive sensitization. Furthermore, patch clamp studies demonstrated that CFA-induced peripheral sensitization primarily affects the excitability of small-diameter DRG neurons. Further studies revealed that inflammatory pain caused by CFA or incubation of DRG with CCL2 mainly affected Ih currents in small-diameter DRG neurons, which were blocked by co-incubation CCR2 antagonist INCB3344 or adenylate cyclase inhibitor SQ22536. Immunohistochemical staining showed that both intraplantar injection of CFA as well as DRG injection of CCL2 resulted in significant upregulation of CCR2+/HCN2+ expression. In conclusion, we suggest in the inflammatory pain state, CCL2 can act on small-diameter DRG neurons, leading to upregulation of HCN2 expression and consequently Ih, which in turn leads to neuronal hyperexcitability. [ABSTRACT FROM AUTHOR]

Summary: The 3′,5′‐cyclic adenosine monophosphate (cAMP) is a versatile second messenger in many mammalian signaling pathways. However, its role in plants remains not well‐recognized. Recent discovery of adenylate cyclase (AC) activity for transport inhibitor response 1/auxin‐signaling F‐box proteins (TIR1/AFB) auxin receptors and the demonstration of its importance for canonical auxin signaling put plant cAMP research back into spotlight. This insight briefly summarizes the well‐established cAMP signaling pathways in mammalian cells and describes the turbulent and controversial history of plant cAMP research highlighting the major progress and the unresolved points. We also briefly review the current paradigm of auxin signaling to provide a background for the discussion on the AC activity of TIR1/AFB auxin receptors and its potential role in transcriptional auxin signaling as well as impact of these discoveries on plant cAMP research in general. [ABSTRACT FROM AUTHOR]

Dopamine receptors (DARs) are important transmembrane receptors responsible for receiving extracellular signals in the DAR-mediated signaling pathway, and are involved in a variety of physiological functions. Herein, the D1 DAR gene from Marsupenaeus japonicus (MjDAD1) was identified and characterized. The protein encoded by MjDAD1 has the typical structure and functional domains of the G-protein coupled receptor family. MjDAD1 expression was significantly upregulated in the gills and hepatopancreas after low temperature stress. Moreover, double-stranded RNA-mediated silencing of MjDAD1 significantly changed the levels of protein kinases (PKA and PKC), second messengers (cyclic AMP (cAMP), cyclic cGMP, calmodulin, and diacyl glycerol), and G-protein effectors (adenylate cyclase and phospholipase C). Furthermore, MjDAD1 silencing increased the apoptosis rate of gill and hepatopancreas cells. Thus, following binding to their specific receptors, G-protein effectors are activated by MjDAD1, leading to DAD1-cAMP/PKA pathway-mediated regulation of caspase-dependent mitochondrial apoptosis. We suggest that MjDAD1 is indispensable for the environmental adaptation of M. japonicus. [ABSTRACT FROM AUTHOR]

The lifecycle of Zymoseptoria tritici requires a carefully regulated asymptomatic phase within the wheat leaf following penetration of the mesophyll via stomata. Here we compare the roles in this process of two key fungal signalling pathways, mutants of which were identified through forward genetics due to their avirulence on wheat. Whole‐genome resequencing of avirulent Z. tritici T‐DNA transformants identified disruptive mutations in ZtBCK1 from the kinase cascade of the cell wall integrity (CWI) pathway, and the adenylate cyclase gene ZtCYR1. Targeted deletion of these genes abolished the pathogenicity of the fungus and led to similar in vitro phenotypes to those associated with disruption of putative downstream kinases, both supporting previous studies and confirming the importance of these pathways in virulence. RNA sequencing was used to investigate the effect of ZtBCK1 and ZtCYR1 deletion on gene expression in both the pathogen and host during infection. ZtBCK1 was found to be required for the adaptation to the host environment, controlling expression of infection‐associated secreted proteins, including known virulence factors. Meanwhile, ZtCYR1 is implicated in controlling the switch to necrotrophy, regulating expression of effectors associated with this transition. This represents the first study to compare the influence of CWI and cAMP signalling on in planta transcription of a fungal plant pathogen, providing insights into their differential regulation of candidate effectors during invasive growth. [ABSTRACT FROM AUTHOR]

Oral tongue squamous cell carcinoma (OTSCC) is a malignant tumor. Recently, studies have found that adenylate cyclase 6 (ADCY6) plays a pivotal role in many lethal tumors formation processes. The role of ADCY6 in OTSCC remains unknown. The expression of ADCY6 in OTSCC tissue samples was detected. The clinical significance of ADCY6 in OTSCC was analyzed by statistical methods. OTSCC cell lines were selected to analyze the biological function of ADCY6. Meanwhile, the effect of ADCY6 on the growth of OTSCC in vivo was explored using subcutaneous tumorigenesis assay. WB assay was used to detect the underlying signaling pathway. Cell function recovery test used to investigate the mechanism of ADCY6‐promoting OTSCC malignant biological behavior via Hippo signaling pathway. We report that ADCY6 was obviously downregulated in OTSCC tissue samples and cell lines. Importantly, lower expression of ADCY6 indicates a poorer prognosis in patients with OTSCC, and its expression is significantly correlated with TNM stage and tumor size. Functionally, forced expression of ADCY6 can significantly inhibit the proliferation, migration, invasion, and promote apoptosis of OTSCC cells. Mechanistically, we demonstrated that ADCY6 upregulation impaired Hippo signaling pathway to reduce the malignant biological behavior of OTSCC. Generally, our findings suggest that ADCY6 suppressed Hippo signaling pathway to regulate malignant biological behavior in OTSCC, which provide new cues for further exploring the mechanism of occurrence and development of OTSCC. [ABSTRACT FROM AUTHOR]

Sickle cell disease (SCD) is an inherited hemoglobinopathy characterized by chronic anemia, intravascular hemolysis, and the occurrence of vaso-occlusive crises due to the mechanical obstruction of the microcirculation by poorly deformable red blood cells (RBCs). RBC deformability is a key factor in the pathogenesis of SCD, and is affected by various factors. In this study, we investigated the effects of adenylyl cyclase (AC) signaling pathway modulation and different phosphodiesterase (PDE) modulatory molecules on the deformability and mechanical stress responses of RBC from SCD patients (HbSS genotype) by applying 5 Pa shear stress with an ektacytometer (LORRCA). We evaluated RBC deformability before and after the application of shear stress. AC stimulation with Forskolin had distinct effects on RBC deformability depending on the application of 5 Pa shear stress. RBC deformability was increased by Forskolin before shear stress application but decreased after 5 Pa shear stress. AC inhibition with SQ22536 and protein kinase A (PKA) inhibition with H89 increased RBC deformability before and after the shear stress application. Non-selective PDE inhibition with Pentoxifylline increased RBC deformability. However, modulation of the different PDE types had distinct effects on RBC deformability, with PDE1 inhibition by Vinpocetine increasing deformability while PDE4 inhibition by Rolipram decreased RBC deformability after the shear stress application. The effects of the drugs varied greatly between patients suggesting some could benefit from one drug while others not. Developing drugs targeting the AC signaling pathway could have clinical applications for SCD, but more researches with larger patient cohorts are needed to identify the differences in the responses of sickle RBCs. [ABSTRACT FROM AUTHOR]

The beta-adrenergic system is a potent stimulus for enhancing cardiac output that may become deleterious when energy metabolism is compromised as in heart failure. We thus examined whether the AMP-activated protein kinase (AMPK) that is activated in response to energy depletion may control the beta-adrenergic pathway. We studied the cardiac response to beta-adrenergic stimulation of AMPKα2-/- mice or to pharmacological AMPK activation on contractile function, calcium current, cAMP content and expression of adenylyl cyclase 5 (AC5), a rate limiting step of the beta-adrenergic pathway. In AMPKα2-/- mice the expression of AC5 (+50%), the dose response curve of left ventricular developed pressure to isoprenaline (p<0.001) or the response to forskolin, an activator of AC (+25%), were significantly increased compared to WT heart. Similarly, the response of L-type calcium current to 3-isobutyl-l-methylxanthine (IBMX), a phosphodiesterase inhibitor was significantly higher in KO (+98%, p<0.01) than WT (+57%) isolated cardiomyocytes. Conversely, pharmacological activation of AMPK by 5-aminoimidazole-4-carboxamide riboside (AICAR) induced a 45% decrease in AC5 expression (p<0.001) and a 40% decrease of cAMP content (P<0.001) as measured by fluorescence resonance energy transfer (FRET) compared to unstimulated rat cardiomyocytes. Finally, in experimental pressure overload-induced cardiac dysfunction, AMPK activation was associated with a decreased expression of AC5 that was blunted in AMPKα2-/- mice. The results show that AMPK activation down-regulates AC5 expression and blunts the beta-adrenergic cascade. This crosstalk between AMPK and beta-adrenergic pathways may participate in a compensatory energy sparing mechanism in dysfunctional myocardium. [ABSTRACT FROM AUTHOR]

We have developed a highly sensitive promoter trap vector system using transposons to generate reporter cells with high efficiency. Using an EGFP/luciferase reporter cell clone responsive to forskolin, which is thought to activate adenylate cyclase, isolated from human chronic myelogenous leukemia cell line K562, we found several compounds unexpectedly caused reporter responses. These included tyrosine kinase inhibitors such as dasatinib and cerdulatinib, which were seemingly unrelated to the forskolin-reactive pathway. To investigate whether any other clones of forskolin-responsive cells would show the same response, nine additional forskolin-responsive clones, each with a unique integration site, were generated and quantitatively evaluated by luciferase assay. The results showed that each clone represented different response patterns to the reactive compounds. Also, it became clear that each of the reactive compounds could be profiled as a unique pattern by the 10 reporter clones. When other TKIs, mainly bcr-abl inhibitors, were evaluated using a more focused set of five reporter clones, they also showed unique profiling. Among them, dasatinib and bosutinib, and imatinib and bafetinib showed homologous profiling. The tyrosine kinase inhibitors mentioned above are approved as anticancer agents, and the system could be used for similarity evaluation, efficacy prediction, etc., in the development of new anticancer agents. [ABSTRACT FROM AUTHOR]

Background and purpose: Alcohol abuse has been associated with erectile dysfunction (ED), but the implicated molecular mechanisms are unresolved. This study analyses the role of alterations in soluble guanylyl cyclase (sGC) in ED. Experimental approach: ED was analysed in adult male C57BL/6J mice subjected to the Chronic Intermittent Ethanol (CIE) paradigm. Erectile function was assessed in anaesthetised mice in vivo by evaluating intracavernosal pressure (ICP) and in vitro in isolated mice corpora cavernosa (CC) mounted in a myograph. Protein expression and reactive oxygen species were analysed by western blot and dihydroethidium staining, respectively. Key results: In CIE mice, we observed a significant decrease in the relaxant response of the CC to stimulation of NO release from nitrergic nerves by electrical field stimulation, to NO release from endothelial cells by acetylcholine, to the PDE5 inhibitor sildenafil, and to the sGC stimulator riociguat. Conversely, the response to the sGC activator cinaciguat, whose action is independent of the oxidation state of sGC, was significantly enhanced in these CC. The responses to adenylyl cyclase stimulation with forskolin were unchanged. We found an increase in reactive oxygen species in the CC from CIE mice as well as an increase in CYP2E1 and NOX2 protein expression. In vivo pre‐treatment with tempol prevented alcohol‐induced erectile dysfunction. Conclusions and implications: Our results demonstrate that alcoholic mice show ED in vitro and in vivo due to an alteration in the redox state of sGC and suggest that sGC activators may be effective in ED associated with alcoholism. [ABSTRACT FROM AUTHOR]

Background: Numerous genes, including SOD1, mutated in familial and sporadic amyotrophic lateral sclerosis (f/sALS) share a role in DNA damage and repair, emphasizing genome disintegration in ALS. One possible outcome of chromosomal instability and repair processes is extrachromosomal circular DNA (eccDNA) formation. Therefore, eccDNA might accumulate in f/sALS with yet unknown function. Methods: We combined rolling circle amplification with linear DNA digestion to purify eccDNA from the cervical spinal cord of 9 co-isogenic symptomatic hSOD1G93A mutants and 10 controls, followed by deep short-read sequencing. We mapped the eccDNAs and performed differential analysis based on the split read signal of the eccDNAs, referred as DifCir, between the ALS and control specimens, to find differentially produced per gene circles (DPpGC) in the two groups. Compared were eccDNA abundances, length distributions and genic profiles. We further assessed proteome alterations in ALS by mass spectrometry, and matched the DPpGCs with differentially expressed proteins (DEPs) in ALS. Additionally, we aligned the ALS-specific DPpGCs to ALS risk gene databases. Results: We found a six-fold enrichment in the number of unique eccDNAs in the genotoxic ALS-model relative to controls. We uncovered a distinct genic circulome profile characterized by 225 up-DPpGCs, i.e., genes that produced more eccDNAs from distinct gene sequences in ALS than under control conditions. The inter-sample recurrence rate was at least 89% for the top 6 up-DPpGCs. ALS proteome analyses revealed 42 corresponding DEPs, of which 19 underlying genes were itemized for an ALS risk in GWAS databases. The up-DPpGCs and their DEP tandems mainly impart neuron-specific functions, and gene set enrichment analyses indicated an overrepresentation of the adenylate cyclase modulating G protein pathway. Conclusions: We prove, for the first time, a significant enrichment of eccDNA in the ALS-affected spinal cord. Our triple circulome, proteome and genome approach provide indication for a potential importance of certain eccDNAs in ALS neurodegeneration and a yet unconsidered role as ALS biomarkers. The related functional pathways might open up new targets for therapeutic intervention. [ABSTRACT FROM AUTHOR]

• Acute morphine stimulation produces analgesic effects by inhibiting adenylyl cyclase activity through D2 receptors. • MORs interact with opioid or non-opioid receptors in the VTA to mediate morphine analgesia and tolerance. • Morphine enhances or inhibits VTA-DA neuron activity in the RMTg-VTA circuit to produce analgesia or tolerance. Morphine has a strong analgesic effect and is suitable for various types of pain, so it is widely used. But long-term usage of morphine can lead to drug tolerance, which limits its clinical application. The complex mechanisms underlying the development of morphine analgesia into tolerance involve multiple nuclei in the brain. Recent studies reveal the signaling at the cellular and molecular levels as well as neural circuits contributing to morphine analgesia and tolerance in the ventral tegmental area (VTA), which is traditionally considered a critical center of opioid reward and addiction. Existing studies show that dopamine receptors and μ-opioid receptors participate in morphine tolerance through the altered activities of dopaminergic and/or non-dopaminergic neurons in the VTA. Several neural circuits related to the VTA are also involved in the regulation of morphine analgesia and the development of drug tolerance. Reviewing specific cellular and molecular targets and related neural circuits may provide novel precautionary strategies for morphine tolerance. [ABSTRACT FROM AUTHOR]

Folding of the Repeats-in-toxin (RTX) domain of the bacterial adenylate cyclase toxin-hemolysin (CyaA) is critical to its toxin activities and the virulence of the whooping cough agent Bordetella pertussis. The RTX domain (RD) contains five RTX blocks (RTX-i to RTX-v) and their folding is driven by the binding of calcium. However, the detailed molecular mechanism via which the folding signal transmits within the five RTX blocks remains unknown. By combining single molecule optical tweezers, protein engineering, and toxin activity assays, here we demonstrate that the folding of the RD follows a strict hierarchy, with the folding starting from its C-terminal block RTXv and proceeding towards the N-terminal RTX-i block sequentially. Our results reveal a strict series, templated folding mechanism, where the folding signal is transmitted along the RD in a series fashion from its C terminus continuously to the N terminus. Due to the series nature of this folding signal transmission pathway, the folding of RD can be disrupted at any given RTX block, rendering the RTX blocks located N-terminally to the disruption site and the acylation region of CyaA unfolded and abolishing CyaA’s toxin activities. Our results reveal key mechanistic insights into the secretion and folding process of CyaA and may open up new potential avenues towards designing new therapeutics to abolish toxin activity of CyaA and combat B. pertussis. [ABSTRACT FROM AUTHOR]

Bivalent epigenomic regulatory domains containing both activating histone 3 lysine 4 (H3K4me3) and repressive lysine 27 (H3K27me3) trimethylation are associated with key developmental genes. These bivalent domains repress transcription in the absence of differentiation signals but maintain regulatory genes in a poised state to allow for timely activation. Previous studies demonstrated that enhancer of zeste homolog 2 (Ezh2), a histone 3 lysine 27 (H3K27) methyltransferase, suppresses osteogenic differentiation and that inhibition of Ezh2 enhances commitment of osteoblast progenitors in vitro and bone formation in vivo. Here, we examined the mechanistic effects of Tazemetostat (EPZ6438), an Food and Drug Administration approved Ezh2 inhibitor for epithelioid sarcoma treatment, because this drug could potentially be repurposed to stimulate osteogenesis for clinical indications. We find that Tazemetostat reduces H3K27me3 marks in bivalent domains in enhancers required for bone formation and stimulates maturation of MC3T3 preosteoblasts. Furthermore, Tazemetostat activates bivalent genes associated with the Wingless/integrated (WNT), adenylyl cyclase (cAMP), and Hedgehog (Hh) signaling pathways based on transcriptomic (RNA-seq) and epigenomic (chromatin immunoprecipitation [ChIP]-seq) data. Functional analyses using selective pathway inhibitors and silencing RNAs demonstrate that the WNT and Hh pathways modulate osteogenic differentiation after Ezh2 inhibition. Strikingly, we show that loss of the Hh-responsive transcriptional regulator Gli1, but not Gli2, synergizes with Tazemetostat to accelerate osteoblast differentiation. These studies establish epigenetic cooperativity of Ezh2, Hh-Gli1 signaling, and bivalent regulatory genes in suppressing osteogenesis. Our findings may have important translational ramifications for anabolic applications requiring bone mass accrual and/or reversal of bone loss. [ABSTRACT FROM AUTHOR]

The melanocortin signaling system consists of the melanocortin peptides, their distinctive receptors, accessory proteins, and endogenous antagonists. Melanocortin peptides are small peptide hormones that have been studied in a variety of physiological and pathological conditions. There are five types of melanocortin receptors, and they are distributed within the central nervous system and in several tissues of the periphery. The G protein–coupled melanocortin receptors typically signal through adenylyl cyclase and other downstream signaling pathways. Depending on the ligand, surface expression of melanocortin receptor, receptor occupancy period, related proteins, the type of cell, and other parameters, the signaling pathways are complicated and pleiotropic. While it is known that all five melanocortin receptors are coupled to Gs, they can also occasionally couple to Gq or Gi. Both direct and indirect neuroprotection are induced by the melanocortin signaling system. Targeting several of the components of the melanocortin signaling system (ligands, receptors, accessory proteins, signaling effectors, and regulators) may provide therapeutic opportunities. Activation of the melanocortin system improves different functional traits in neurodegenerative diseases. There is a potential for additional melanocortin system interventions by interfering with dimerization or dissociation. This review aims to discuss the melanocortin receptor signaling system and its role in neuroprotection, as well as its therapeutic potential. [ABSTRACT FROM AUTHOR]

Soluble adenylyl cyclase (sAC)‐derived cAMP regulates various cellular processes; however, the regulatory landscape mediating sAC protein levels remains underexplored. We consistently observed a 85 kD (sAC85) or 75 kD (sAC75) sAC protein band under glucose‐sufficient or glucose‐deprived states, respectively, in H69 cholangiocytes by immunoblotting. Deglycosylation by PNGase‐F demonstrated that both sAC75 and sAC85 are N‐linked glycosylated proteins with the same polypeptide backbone. Deglycosylation with Endo‐H further revealed that sAC75 and sAC85 carry distinct sugar chains. We observed release of N‐linked glycosylated sAC (sACEV) in extracellular vesicles under conditions that support intracellular sAC85 (glucose‐sufficient) as opposed to sAC75 (glucose‐deprived) conditions. Consistently, disrupting the vesicular machinery affects the maturation of intracellular sAC and inhibits the release of sACEV into extracellular vesicles. The intracellular turnover of sAC85 is extremely short (t1/2 ~30 min) and release of sACEV in the medium was detected within 3 h. Our observations support the maturation and trafficking in cholangiocytes of an N‐linked glycosylated sAC isoform that is rapidly released into extracellular vesicles. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors in trigeminal ganglion (TG) neurons are often associated with sensory mechanisms, including nociception. We have previously reported the expression of P2Y12 receptors, which are Gi protein-coupled receptors, in TG cells. Activating P2Y12 receptors decreased the intracellular free Ca2+ concentration ([Ca2+]i). This indicated that intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) levels can mediate Ca2+ signaling in TG cells. Here, we report more extensive-expression patterns of Gs protein-coupled receptors in primary cultured TG neurons isolated from 7-day-old newborn Wistar rats and further examine the roles of these receptors in cAMP signaling using the BacMam sensor in these neurons. To identify TG neurons, we also measured [Ca2+]i using fura-2 in TG cells and measured intracellular cAMP levels. TG neurons were positive for Gαs protein-coupled receptors, beta-2 adrenergic (β2), calcitonin gene-related peptide (CGRP), adenosine A2A (A2A), dopamine 1 (D1), prostaglandin I2 (IP), and 5-hydroxytriptamine 4 (5-HT4) receptor. Application of forskolin (FSK), an activator of adenylyl cyclase, transiently increased intracellular cAMP levels in TG neurons. The application of a phosphodiesterase inhibitor augmented the FSK-elicited intracellular cAMP level increase. These increases were significantly suppressed by the application of SQ22536, an adenylyl cyclase inhibitor, in TG neurons. Application of agonists for β2, CGRP, A2A, D1-like, IP, and 5-HT4 receptors increased intracellular cAMP levels. These increases were SQ22536-sensitive. These results suggested that TG neurons express β2, CGRP, A2A, D1, IP, and 5-HT4 receptors, and the activations of these Gαs protein-coupled receptors increase intracellular cAMP levels by activating adenylyl cyclase. [ABSTRACT FROM AUTHOR]

CFTR (cystic fibrosis transmembrane conductance regulator) is a tightly regulated anion channel that mediates chloride and bicarbonate conductance in many epithelia and in other tissues, but whether its regulation varies depending on the cell type has not been investigated. Epithelial CFTR expression is highest in rare cells called ionocytes. We studied CFTR regulation in control and ionocyteenriched cultures by transducing bronchial basal cells with adenoviruses that encode only eGFP or FOXI1 (forkhead box I1) 1 eGFP as separate polypeptides. FOXI1 dramatically increased the number of transcripts for ionocyte markers ASCL3 (Achaete-Scute Family BHLH Transcription Factor 3), BSND, ATP6V1G3, ATP6V0D2, KCNMA1, and CFTR without altering those for secretory (SCGB1A1), basal (KRT5, KRT6, TP63), goblet (MUC5AC), or ciliated (FOXJ1) cells. The number of cells displaying strong FOXI1 expression was increased 7-fold, and there was no evidence for a broad increase in background immunofluorescence. Total CFTR mRNA and protein levels increased 10-fold and 2.5-fold, respectively. Ionocyte-enriched cultures displayed elevated basal current, increased adenylyl cyclase 5 expression, and tonic suppression of CFTR activity by the phosphodiesterase PDE1C, which has not been shown previously to regulate CFTR activity. The results indicate that CFTR regulation depends on cell type and identifies PDE1C as a potential target for therapeutics that aim to increase CFTR function specifically in ionocytes. [ABSTRACT FROM AUTHOR]

Following the neutral results of the dal-OUTCOMES trial, a genome-wide study identified the rs1967309 variant in the adenylate cyclase type 9 (ADCY9) gene on chromosome 16 as being associated with the risk of future cardiovascular events only in subjects taking dalcetrapib, a CETP (cholesterol ester transfer protein) modulator. Homozygotes for the minor A allele (AA) were protected from recurrent cardiovascular events when treated with dalcetrapib, while homozygotes for the major G allele (GG) had increased risk. Here, we present the current state of knowledge regarding the impact of rs1967309 in ADCY9 on clinical observations and biomarkers in dalcetrapib trials and the effects of mouse ADCY9 gene inactivation on cardiovascular physiology. Finally, we present our current model of the interaction between dalcetrapib and ADCY9 gene variants in the arterial wall macrophage, based on the intracellular role of CETP in the transfer of complex lipids from endoplasmic reticulum membranes to lipid droplets. Briefly, the concept is that dalcetrapib would inhibit CETP-mediated transfer of cholesteryl esters, resulting in a progressive inhibition of cholesteryl ester synthesis and free cholesterol accumulation in the endoplasmic reticulum. Reduced ADCY9 activity, by paradoxically leading to higher cyclic AMP levels and in turn increased cellular cholesterol efflux, could impart cardiovascular protection in rs1967309 AA patients. The ongoing dal-GenE trial recruited 6145 patients with the protective AA genotype and will provide a definitive answer to whether dalcetrapib will be protective in this population.

This article, titled "Editorial: Color change: neural and hormonal control of pigmentation," discusses the neural and hormonal control of pigmentation in various organisms. The pigment system, found in vertebrates, crustaceans, and cephalopods, is essential for survival and adaptation, allowing organisms to respond to internal and external stimuli. Different types of chromatophores, specialized cells that contain pigments, are responsible for producing various colors. The behavior of chromatophores is controlled by the nervous system, hormonal system, or both. Hormones and neurotransmitters regulate chromatophore responses, inducing pigment aggregation or dispersal. The article also explores the different types of chromatophores and their optical properties. Hormones such as a-MSH, MCH, catecholamines, and melatonin play important roles in hormonal regulation of pigment cells. The article concludes by highlighting the complexity of chromatic adaptation and color patterns at the cellular and molecular levels. [Extracted from the article]

The phytopathogenic fungus Fusarium fujikuroi has a rich secondary metabolism which includes the synthesis of very different metabolites in response to diverse environmental cues, such as light or nitrogen. Here, we focused our attention on fusarins, a class of mycotoxins whose synthesis is downregulated by nitrogen starvation. Previous data showed that mutants of genes involved in carotenoid regulation (carS, encoding a RING finger protein repressor), light detection (wcoA, White Collar photoreceptor), and cAMP signaling (AcyA, adenylate cyclase) affect the synthesis of different metabolites. We studied the effect of these mutations on fusarin production and the expression of the fus1 gene, which encodes the key polyketide synthase of the pathway. We found that the three proteins are positive regulators of fusarin synthesis, especially WcoA and AcyA, linking light regulation to cAMP signaling. Genes for two other photoreceptors, the cryptochrome CryD and the Vivid flavoprotein VvdA, were not involved in fusarin regulation. In most cases, there was a correspondence between fusarin production and fus1 mRNA, indicating that regulation is mainly exerted at the transcriptional level. We conclude that fusarin synthesis is subject to a complex control involving regulators from different signaling pathways.

Background: Adenylate cyclases (ACs) generate the second messenger cyclic AMP (cAMP), which is found in all domains of life and is involved in the regulation of various cell physiological and metabolic processes. In the plant symbiotic bacterium Sinorhizobium meliloti, synthesis of cAMP by the membrane-bound AC CyaC responds to the redox state of the respiratory chain and the respiratory quinones. However, nothing is known about the signaling cascade that is initiated by cAMP produced by CyaC. Results: Here, the CRP-like transcriptional regulator Clr and the TetR-like regulator CycR (TR01819 protein) were identified to interact with CyaC using the bacterial two-hybrid system (BACTH), co-sedimentation assays, and surface plasmon resonance spectroscopy. Interaction of CycR with Clr, and of CyaC with Clr requires the presence of cAMP and of ATP, respectively, whereas that of CyaC with CycR was independent of the nucleotides. Conclusion: The data implicate a ternary CyaC×CycR×cAMP-Clr complex, functioning as a specific signaling cascade which is formed after activation of CyaC and synthesis of cAMP. cAMP-Clr is thought to work in complex with CycR to regulate a subset of genes of the cAMP-Clr regulon in S. meliloti. [ABSTRACT FROM AUTHOR]

[Display omitted] • GPR81 is a widely distributed receptor that binds L-lactate and acts as a metabolic sensor. • In the brain, GPR81 couples energy metabolism, synaptic activity, and blood flow. • We propose GPR81 as a potential target in cerebral ischemia. GPR81 is a G-protein coupled receptor (GPCR) discovered in 2001, but deorphanized only 7 years later, when its affinity for lactate as an endogenous ligand was demonstrated. More recently, GPR81 expression and distribution in the brain were also confirmed and the function of lactate as a volume transmitter has been suggested since then. These findings shed light on a new function of lactate acting as a signaling molecule in the central nervous system, in addition to its well-known role as a metabolic fuel for neurons. GPR81 seems to act as a metabolic sensor, coupling energy metabolism, synaptic activity, and blood flow. Activation of this receptor leads to G i -mediated downregulation of adenylyl cyclase and subsequent reduction in cAMP levels, regulating several downstream pathways. Recent studies have also suggested the potential role of lactate as a neuroprotective agent, mainly under brain ischemic conditions. This effect is usually attributed to the metabolic role of lactate, but the underlying mechanisms need further investigation and could be related to lactate signaling via GPR81. The activation of GPR81 showed promising results for neuroprotection: it modulates many processes involved in the pathophysiology of ischemia. In this review, we summarize the history of GPR81, starting with its deorphanization; then, we discuss GPR81 expression and distribution, signaling transduction cascades, and neuroprotective roles. Lastly, we propose GPR81 as a potential target for the treatment of cerebral ischemia. [ABSTRACT FROM AUTHOR]

Introduction: Corni Fructus (CF) is a Chinese herbal medicine used for medicinal and dietary purposes. It is available commercially in two main forms: raw CF (unprocessed CF) and wine-processed CF. Clinical observations have indicated that wine-processed CF exhibits superior hypoglycemic activity compared to its raw counterpart. However, the mechanisms responsible for this improvement are not well understood. Methods: To address this gap in knowledge, we conducted metabolomics analysis using ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UPLC-QTOF-MS) to compare the chemical composition of raw CF and wine-processed CF. Subsequently, network analysis, along with immunofluorescence assays, was employed to elucidate the potential targets and mechanisms underlying the hypoglycemic effects of metabolites in CF. Results: Our results revealed significant compositional differences between raw CF and wine-processed CF, identifying 34 potential markers for distinguishing between the two forms of CF. Notably, wine processing led to a marked decrease in iridoid glycosides and flavonoid glycosides, which are abundant in raw CF. Network analysis predictions provided clues that eight compounds might serve as hypoglycemic metabolites of CF, and glucokinase (GCK) and adenylate cyclase (ADCYs) were speculated as possible key targets responsible for the hypoglycemic effects of CF. Immunofluorescence assays confirmed that oleanolic acid and ursolic acid, two bioactive compounds present in CF, significantly upregulated the expression of GCK and ADCYs in the HepG2 cell model. Discussion: These findings support the notion that CF exerted hypoglycemic activity via multiple components and targets, shedding light on the impact of processing methods on the chemical composition and hypoglycemic activity of Chinese herbal medicine. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem cells (iPSCs) have enabled the generation of various difficult-to-access cell types such as human nociceptors. A key challenge associated with human iPSC-derived nociceptors (hiPSCdNs) is their prolonged functional maturation. While numerous studies have addressed the expression of classic neuronal markers and ion channels in hiPSCdNs, the temporal development of key signaling cascades regulating nociceptor activity has remained largely unexplored. In this study, we used an immunocytochemical high-content imaging approach alongside electrophysiological staging to assess metabotropic and ionotropic signaling of large scale–generated hiPSCdNs across 70 days of in vitro differentiation. During this period, the resting membrane potential became more hyperpolarized, while rheobase, action potential peak amplitude, and membrane capacitance increased. After 70 days, hiPSCdNs exhibited robust physiological responses induced by GABA, pH shift, ATP, and capsaicin. Direct activation of protein kinase A type II (PKA-II) through adenylyl cyclase stimulation with forskolin resulted in PKA-II activation at all time points. Depolarization-induced activation of PKA-II emerged after 35 days of differentiation. However, effective inhibition of forskolin-induced PKA-II activation by opioid receptor agonists required 70 days of in vitro differentiation. Our results identify a pronounced time difference between early expression of functionally important ion channels and emergence of regulatory metabotropic sensitizing and desensitizing signaling only at advanced stages of in vitro cultivation, suggesting an independent regulation of ionotropic and metabotropic signaling. These data are relevant for devising future studies into the development and regulation of human nociceptor function and for defining time windows suitable for hiPSCdN-based drug discovery. [ABSTRACT FROM AUTHOR]

Clonostachys rosea is an excellent biocontrol fungus against numerous fungal plant pathogens. The cAMP signaling pathway is a crucial signal transduction pathway in fungi. To date, the role of the cAMP signaling pathway in C. rosea mycoparasitism remains unknown. An adenylate cyclase-encoding gene, crac (an important component of the cAMP signaling pathway), was previously screened from C. rosea 67-1, and its expression level was dramatically upregulated during the C. rosea mycoparasitization of the sclerotia of Sclerotinia sclerotiorum. In this study, the function of crac in C. rosea mycoparasitism was explored through gene knockout and complementation. The obtained results show that the deletion of crac influenced the growth rate and colony morphology of C. rosea, as well as the tolerance to NaCl and H2O2 stress. The mycoparasitic effects on the sclerotia of S. sclerotiorum and the biocontrol capacity on soybean Sclerotinia stem rot in ∆crac-6 and ∆crac-13 were both attenuated compared with that of the wild-type strain and complementation transformants. To understand the regulatory mechanism of crac during C. rosea mycoparasitism, transcriptomic analysis was conducted between the wild-type strain and knockout mutant. A number of biocontrol-related genes, including genes encoding cell wall-degrading enzymes and transporters, were significantly differentially expressed during C. rosea mycoparasitism, suggesting that crac may be involved in C. rosea mycoparasitism by regulating the expression of these DEGs. These findings provide insight for further exploring the molecular mechanism of C. rosea mycoparasitism. [ABSTRACT FROM AUTHOR]

Photoactivated adenylate cyclases (PACs) are light activated enzymes that combine blue light sensing capacity with the ability to convert ATP to cAMP and pyrophosphate (PPi) in a light-dependent manner. In most of the known PACs blue light regulation is provided by a blue light sensing domain using flavin which undergoes a structural reorganization after bluelight absorption. This minor structural change then is translated toward the C-terminal of the protein, inducing a larger conformational change that results in the ATP conversion to cAMP. As cAMP is a key second messenger in numerous signal transduction pathways regulating various cellular functions, PACs are of great interest in optogenetic studies. The optimal optogenetic device must be “silent” in the dark and highly responsive upon light illumination. PAC from Oscillatoria acuminata is a very good candidate as its basal activity is very small in the dark and the conversion rates increase 20-fold upon light illumination. We studied the effect of replacing D67 to N, in the blue light using flavin domain. This mutation was found to accelerate the primary electron transfer process in the photosensing domain of the protein, as has been predicted. Furthermore, it resulted in a longer lived signaling state, which was formed with a lower quantum yield. Our studies show that the overall effects of the D67N mutation lead to a slightly higher conversion of ATP to cAMP, which points in the direction that by fine tuning the kinetic properties more responsive PACs and optogenetic devices can be generated. [ABSTRACT FROM AUTHOR]

The acylated Repeats in ToXins (RTX) leukotoxins, the adenylate cyclase toxin (CyaA) or α-hemolysin (HlyA), bind β2 integrins of leukocytes but also penetrate cells lacking these receptors. We show that the indoles of conserved tryptophans in the acylated segments, W876 of CyaA and W579 of HlyA, are crucial for β2 integrin–independent membrane penetration. Substitutions of W876 by aliphatic or aromatic residues did not affect acylation, folding, or the activities of CyaA W876L/F/Y variants on cells expressing high amounts of the β2 integrin CR3. However, toxin activity of CyaA W876L/F/Y on cells lacking CR3 was strongly impaired. Similarly, a W579L substitution selectively reduced HlyA W579L cytotoxicity towards cells lacking β2 integrins. Intriguingly, the W876L/F/Y substitutions increased the thermal stability (Tm) of CyaA by 4 to 8°C but locally enhanced the accessibility to deuteration of the hydrophobic segment and of the interface of the two acylated loops. W876Q substitution (showing no increase in Tm), or combination of W876F with a cavity-filling V822M substitution (this combination decreasing the Tm closer to that of CyaA), yielded a milder defect of toxin activity on erythrocytes lacking CR3. Furthermore, the activity of CyaA on erythrocytes was also selectively impaired when the interaction of the pyrrolidine of P848 with the indole of W876 was ablated. Hence, the bulky indoles of residues W876 of CyaA, or W579 of HlyA, rule the local positioning of the acylated loops and enable a membrane-penetrating conformation in the absence of RTX toxin docking onto the cell membrane by β2 integrins. [ABSTRACT FROM AUTHOR]

Under conditions of neurodegeneration modeled in vitro by the β-amyloid peptide-(25-35) fragment (Aβ25-35), we studied the role of individual links of cAMP-dependent intracellular signaling pathways in determining the proliferation and differentiation status of neural stem cells (NSCs) and colony-stimulating activity of supernatants from neuroglial cells. The important role of intracellular cAMP and PKA in the inhibition of the progression of the NSC cell cycle and stimulation of the process of their specialization induced by Aβ25-35 was found. The selective ability of PKA to block the production of factors constituting colony-stimulating activity by neuroglial cells under conditions of their cultivation in vitro with a neurotoxic agent was revealed. Our results suggests that inhibitors of adenylate cyclase and PKA can increase the degree of implementation of the growth potential of NSCs and conjugation of the processes of their proliferation and differentiation in Alzheimer's disease. At the same time, selective PKA blockers can also induce the production of NSC-stimulating factors by neuroglial cells. [ABSTRACT FROM AUTHOR]

Sinoatrial node (SAN) beating interval variability (BIV) and the average beating interval (BI) are regulated by a coupled-clock system, driven by Ca2+-calmodulin activated adenylyl cyclase, cAMP, and downstream PKA signaling. Reduced responsiveness of the BI and BIV to submaximal, [X]50, β-adrenergic receptor (β-AR) stimulation, and phosphodiesterase inhibition (PDEI) have been documented in aged SAN tissue, whereas the maximal responses, [X]max, do not differ by age. To determine whether age-associated dysfunction in cAMP signaling leads to altered responsiveness of BI and BIV, we measured cAMP levels and BI in adult (2–4 months n = 27) and aged (22–26 months n = 25) C57/BL6 mouse SAN tissue in control and in response to β-AR or PDEI at X50 and [X]max. Both cAMP and average BI in adult SAN were reduced at X50, whereas cAMP and BI at Xmax did not differ by age. cAMP levels and average BI were correlated both within and between adult and aged SAN. BIV parameters in long- and short-range terms were correlated with cAMP levels for adult SAN. However, due to reduced cAMP within aged tissues at [X]50, these correlations were diminished in advanced age. Thus, cAMP level generated by the coupled clock mechanisms is tightly linked to average BI. Reduced cAMP level at X50 in aged SAN explains the reduced responsiveness of the BI and BIV to β-AR stimulation and PDEI. [ABSTRACT FROM AUTHOR]

Widespread release of norepinephrine (NE) throughout the forebrain fosters learning and memory via adrenergic receptor (AR) signaling, but the molecular mechanisms are largely unknown. The β² AR and its downstream effectors, the trimeric stimulatory Gs-protein, adenylyl cyclase (AC), and the cAMP-dependent protein kinase A (PKA), form a unique signaling complex with the L-type Ca2+ channel (LTCC) CaV1.2. Phosphorylation of CaV1.2 by PKA on Ser1928 is required for the upregulation of Ca2+ influx on β2 AR stimulation and long-term potentiation induced by prolonged theta-tetanus (PTT-LTP) but not LTP induced by two 1-s-long 100-Hz tetani. However, the function of Ser1928 phosphorylation in vivo is unknown. Here, we show that S1928A knock-in (KI) mice of both sexes, which lack PTT-LTP, express deficiencies during initial consolidation of spatial memory. Especially striking is the effect of this mutation on cognitive flexibility as tested by reversal learning. Mechanistically, long-term depression (LTD) has been implicated in reversal learning. It is abrogated in male and female S1928A knock-in mice and by β2 AR antagonists and peptides that displace β2 AR from CaV1.2. This work identifies CaV1.2 as a critical molecular locus that regulates synaptic plasticity, spatial memory and its reversal, and LTD. [ABSTRACT FROM AUTHOR]

Cyclic AMP signalling in trypanosomes differs from most eukaryotes due to absence of known cAMP effectors and cAMP independence of PKA. We have previously identified four genes from a genome-wide RNAi screen for resistance to the cAMP phosphodiesterase (PDE) inhibitor NPD-001. The genes were named cAMP Response Protein (CARP) 1 through 4. Here, we report an additional six CARP candidate genes from the original sample, after deep sequencing of the RNA interference target pool retrieved after NPD-001 selection (RIT-seq). The resistance phenotypes were confirmed by individual RNAi knockdown. Highest level of resistance to NPD-001, approximately 17-fold, was seen for knockdown of CARP7 (Tb927.7.4510). CARP1 and CARP11 contain predicted cyclic AMP binding domains and bind cAMP as evidenced by capture and competition on immobilised cAMP. CARP orthologues are strongly enriched in kinetoplastid species, and CARP3 and CARP11 are unique to Trypanosoma. Localization data and/or domain architecture of all CARPs predict association with the T. brucei flagellum. This suggests a crucial role of cAMP in flagellar function, in line with the cell division phenotype caused by high cAMP and the known role of the flagellum for cytokinesis. The CARP collection is a resource for discovery of unusual cAMP pathways and flagellar biology. [ABSTRACT FROM AUTHOR]

The type 1 and 8 adenylyl cyclase (ADCY1 and ADCY8) exclusively account for Ca2+-stimulated cyclic AMP (cAMP) production and regulate activity-dependent synaptic modification. In this study, we examined distinct forms of synaptic plasticity in the hippocampus of Adcy1-/- and Adcy8-/- mice. We found that, at the Schaer collateral-CA1 synapses, while the Adcy8-/- mice displayed normal long-term potentiation (LTP) following various induction protocols with highfrequency stimulation (HFS), the Adcy1-/- mice showed protocol-dependent deficits in LTP. We also found that long-term depression (LTD) requires ADCY1 but not ADCY8. Interestingly, both Adcy1-/- and Adcy8-/- mice showed defective synaptic depotentiation (i.e., activity-dependent reversal of LTP); the deficits in Adcy8-/- mice were dependent on the induction protocol. Examination of spatial memory found that ADCY1 is required for the formation of both initial and reversal memory. ADCY8 is only required for reversal memory formation. These data demonstrate that ADCY1 and ADCY8 play distinct roles in regulating synaptic and cognitive flexibility that involves bidirectional modification of synaptic function. [ABSTRACT FROM AUTHOR]

Adenylyl cyclase (AC) is the vital enzyme for generating 3',5'-cyclic adenosine monophosphate, an important signaling molecule with profound nutritional and medicinal values. However, merely, a dozen of AC proteins have been reported in plants so far. Here, a protein annotated as triphosphate tunnel metalloenzyme (PbrTTM1) in pear, the important worldwide fruit plant, was firstly identified to possess AC activity with both in vivo and in vitro methods. It exhibited a relatively low AC activity but was capable of complementing AC functional deficiencies in the E. coli SP850 strain. Its protein conformation and potential catalytic mechanism were analyzed by means of biocomputing. The active site of PbrTTM1 is a closed tunnel constructed by nine antiparallel b-folds surrounded with seven helices. Inside the tunnel, the charged residues were possibly involved in the catalytic process by coordinating with divalent cation and ligand. The hydrolysis activity of PbrTTM1 was tested as well. Compared to the much higher capacity of hydrolyzing, the AC activity of PbrTTM1 tends to be a moonlight function. Through a comparison of protein structures in various plant TTMs, it is reasonable to speculate that many plant TTMs might possess AC activity as a form of moonlighting enzyme function. [ABSTRACT FROM AUTHOR]

The melanocortin-4 receptor (MC4R) plays a critical role in regulating energy homeostasis. Studies on obesogenic human MC4R (hMC4R) variants have not yet revealed how hMC4R maintains body weight. Here, we identified a signaling profile for obesogenic constitutively active H76R and L250Q hMC4R variants transfected in HEK293 cells that included constitutive activity for adenylyl cyclase (AC), cyclic adenosine monophosphate (cAMP) response element (CRE)-driven transcription, and calcium mobilization but not phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) activity. Importantly, the signaling profile included impaired α-melanocyte-stimulating hormone-induced CREdriven transcription but not impaired α-melanocyte-stimulating hormone-induced AC, calcium, or pERK1/2. This profile was not observed for transfected H158R, a constitutively active hMC4R variant associated with overweight but not obesity. We concluded that there is potential for α-melanocyte-stimulating hormone-induced CRE-driven transcription in HEK293 cells transfected with obesogenic hMC4R variants to be the key predictive tool for determining whether they exhibit loss of function. Furthermore, in vivo, α-melanocyte-stimulating hormone-induced hMC4R CRE-driven transcription may be key for maintaining body weight. [ABSTRACT FROM AUTHOR]

In Paramecium, a mechanical stimulus applied to the posterior portion of the cell causes a transient increase in membrane permeability to potassium ions, transiently rendering the membrane in a hyperpolarized state. Hyperpolarization causes a transient increase in Cyclic adenosine monophosphate (cAMP) concentration in the cilia, resulting in a transient fast-forward swimming of the cell. Schultz and coworkers (1992) reported that a unique adenylate cyclase (AC)-coupled potassium channel is involved in the reaction underlying this response, which is known as the “escape response.” However, the AC responsible for this reaction remains to be identified. Moreover, the molecular linkage between mechanoreception and AC activation has not been elucidated adequately. Currently, we can perform an efficient and simple gene-knockdown technique in Paramecium using RNA interference (RNAi). Paramecium is one of the several model organisms for which whole-genome sequences have been elucidated. The RNAi technique can be applied to whole genome sequences derived from the Paramecium database (ParameciumDB) to investigate the types of proteins that elicit specific biological responses and compare them with those of other model organisms. In this review, we describe the applications of the RNAi technique in elucidating the molecular mechanism underlying the escape response and identifying the AC involved in this reaction. The findings of this study highlight the advantages of the RNAi technique and ParameciumDB. [ABSTRACT FROM AUTHOR]