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The evolutionarily conserved soluble adenylyl cyclase (sAC, ADCY10) mediates cAMP signaling exclusively in intracellular compartments. Because sAC activity is sensitive to local concentrations of ATP, bicarbonate, and free Ca 2+ , sAC is potentially an important metabolic sensor. Nonetheless, little is known about how sAC regulates energy metabolism in intact cells. In this study, we demonstrated that both pharmacological and genetic suppression of sAC resulted in increased lactate secretion and decreased pyruvate secretion in multiple cell lines and primary cultures of mouse hepatocytes and cholangiocytes. The increased extracellular lactate-to-pyruvate ratio upon sAC suppression reflected an increased cytosolic free [NADH]/[NAD + ] ratio, which was corroborated by using the NADH/NAD + redox biosensor Peredox-mCherry. Mechanistic studies in permeabilized HepG2 cells showed that sAC inhibition specifically suppressed complex I of the mitochondrial respiratory chain. A survey of cAMP effectors revealed that only selective inhibition of exchange protein activated by cAMP 1 (Epac1), but not protein kinase A (PKA) or Epac2, suppressed complex I-dependent respiration and significantly increased the cytosolic NADH/NAD + redox state. Analysis of the ATP production rate and the adenylate energy charge showed that inhibiting sAC reciprocally affects ATP production by glycolysis and oxidative phosphorylation while maintaining cellular energy homeostasis. In conclusion, our study shows that, via the regulation of complex I-dependent mitochondrial respiration, sAC-Epac1 signaling regulates the cytosolic NADH/NAD + redox state, and coordinates oxidative phosphorylation and glycolysis to maintain cellular energy homeostasis. As such, sAC is effectively a bioenergetic switch between aerobic glycolysis and oxidative phosphorylation at the post-translational level., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Paralichthys olivaceus is the kind of cold-water benthic marine fish. In the early stages of development, the symmetrical juveniles transform into an asymmetrical body shape through metamorphosis for adapting benthic life. After that, one side of the fish body is attached to the ground, and the eyes turn to the opposite side which is called ocular side. The body color also appears asymmetry. The skin on the ocular side is dark brown, and the skin on the blind side is white without pigmentation. Pseudo-albinism and hypermelanosis have been considered distinct body color disorders in flatfish. Pseudo-albinism and hypermelanosis in Paralichthys olivaceus are due to abnormal or uneven pigment distribution, due to the interaction of hereditary and environmental factors, rather than a single-nucleotide mutation of a specific gene. Here, we report three single-nucleotide polymorphisms (SNPs) responsible for both pseudo-albinism and hypermelanosis, which are located on two body color-related genes involved in melanogenesis-related pathways. c.2440C>A (P. V605I) and c.2271-96T>C are located on the Inositol 1,4,5-trisphosphate receptor type 2-like (ITPR2) (Gene ID: 109624047), they are located in exon 16 and the non-coding region, respectively, and c.2406C>A (P.H798N) is located in exon 13 of the adenylate cyclase type 6-like (AC6) gene(Gene ID: 109630770). ITPR2 and AC6 expression, which both participate in the thyroid hormone synthesis pathway associated with pseudo-albinism and hypermelanosis in P. olivaceus, were also investigated using qRT-PCR. In hypermelanotic fish, there were relatively higher levels of expression in ITPR2 and AC6 mRNA of hyper-pigmented skin of blind side than that of non-pigmented skin on the blind side and pigmented skin on the ocular side, while in pseudo-albino fish, expression level of ITPR2 and AC6 mRNA in pigmented skin of ocular side was significantly higher than that in non-pigmented skin both ocular and blind side. The results indicated that the expression of the two genes in abnormal parts of body color is positively correlated with pigmentation, suggesting that the influence of abnormal expression of two genes on the pigmentation in abnormal parts of body color deserves further study.

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.

Apelin receptor (APJ) activation by apelin-13 (APLN-13) engages both Gαi proteins and β-arrestins, stimulating distinct intracellular pathways and triggering physiological responses like enhanced cardiac contractility. Substituting the C-terminal phenylalanine of APLN-13 with α-methyl-l-phenylalanine [(l-α-Me)Phe] or p-benzoyl-l-phenylalanine (Bpa) generates biased analogs inducing APJ functional selectivity toward Gαi proteins. Using these original analogs, we proposed to investigate how the canonical Gαi signaling of APJ regulates the cardiac function and to assess their therapeutic impact in a rat model of isoproterenol-induced myocardial dysfunction. In vivo and ex vivo infusions of either Bpa or (l-α-Me)Phe analogs failed to enhance rats' left ventricular (LV) contractility compared with APLN-13. Inhibition of Gαi with pertussis toxin injection optimized the cardiotropic effect of APLN-13 and revealed the inotropic impact of Bpa. Moreover, both APLN-13 and Bpa efficiently limited the forskolin-induced and PKA-dependent phosphorylation of phospholamban at the Ser16 in neonatal rat ventricular myocytes. However, only Bpa significantly reduced the inotropic effect of forskolin infusion in isolated-perfused heart, highlighting its efficient bias toward Gαi. Compared with APLN-13, Bpa also markedly improved isoproterenol-induced myocardial systolic and diastolic dysfunctions. Bpa prevented cardiac weight increase, normalized both ANP and BNP mRNA expressions, and decreased LV fibrosis in isoproterenol-treated rats. Our results show that APJ-driven Gαi/adenylyl cyclase signaling is functional in cardiomyocytes and acts as negative feedback of the APLN-APJ-dependent inotropic response. Biased APJ signaling toward Gαi over the β-arrestin pathway offers a promising strategy in the treatment of cardiovascular diseases related to myocardial hypertrophy and high catecholamine levels. NEW & NOTEWORTHY By using more potent Gαi-biased APJ agonists that strongly inhibit cAMP production, these data point to the negative inotropic effect of APJ-mediated Gαi signaling in the heart and highlight the potential protective impact of APJ-dependent Gαi signaling in cardiovascular diseases associated with left ventricular hypertrophy.

Rationale: Ferroptosis, a newly identified form of regulated cell death, can be induced following the inhibition of cystine-glutamate antiporter system X C - because of the impaired uptake of cystine. However, the outcome following the accumulation of endogenous glutamate in lung adenocarcinoma (LUAD) has not yet been determined. Yes-associated protein (YAP) is sustained by the hexosamine biosynthesis pathway (HBP)-dependent O-linked beta-N-acetylglucosaminylation (O-GlcNAcylation), and glutamine-fructose-6-phosphate transaminase (GFPT1), the rate-limiting enzyme of the HBP, can be phosphorylated and inhibited by adenylyl cyclase (ADCY)-mediated activation of protein kinase A (PKA). However, whether accumulated endogenous glutamate determines ferroptosis sensitivity by influencing the ADCY/PKA/HBP/YAP axis in LUAD cells is not understood. Methods: Cell viability, cell death and the generation of lipid reactive oxygen species (ROS) and malondialdehyde (MDA) were measured to evaluate the responses to the induction of ferroptosis following the inhibition of system X C - . Tandem mass tags (TMTs) were employed to explore potential factors critical for the ferroptosis sensitivity of LUAD cells. Immunoblotting (IB) and quantitative RT-PCR (qPCR) were used to analyze protein and mRNA expression. Co-immunoprecipitation (co-IP) assays were performed to identify protein-protein interactions and posttranslational modifications. Metabolite levels were measured using the appropriate kits. Transcriptional regulation was evaluated using a luciferase reporter assay, chromatin immunoprecipitation (ChIP), and electrophoretic mobility shift assay (EMSA). Drug administration and limiting dilution cell transplantation were performed with cell-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models. The associations among clinical outcome, drug efficacy and ADCY10 expression were determined based on data from patients who underwent curative surgery and evaluated with patient-derived primary LUAD cells and tissues. Results: The accumulation of endogenous glutamate following system X C - inhibition has been shown to determine ferroptosis sensitivity by suppressing YAP in LUAD cells. YAP O-GlcNAcylation and expression cannot be sustained in LUAD cells upon impairment of GFPT1. Thus, Hippo pathway-like phosphorylation and ubiquitination of YAP are enhanced. ADCY10 acts as a key downstream target and diversifies the effects of glutamate on the PKA-dependent suppression of GFPT1. We also discovered that the protumorigenic and proferroptotic effects of ADCY10 are mediated separately. Advanced-stage LUADs with high ADCY10 expression are sensitive to ferroptosis. Moreover, LUAD cells with acquired therapy resistance are also prone to higher ADCY10 expression and are more likely to respond to ferroptosis. Finally, a varying degree of secondary labile iron increase is caused by the failure to sustain YAP-stimulated transcriptional compensation for ferritin at later stages further explains why ferroptosis sensitivity varies among LUAD cells. Conclusions: Endogenous glutamate is critical for ferroptosis sensitivity following the inhibition of system X C - in LUAD cells, and ferroptosis-based treatment is a good choice for LUAD patients with later-stage and/or therapy-resistant tumors., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Herein we hypothesized that DPP10-AS1 could affect the development of colon cancer via the interaction with miR-127-3p and adenylate cyclase 1 (ADCY1). After sorting of CD133 positive cells, sphere formation, colony formation, proliferation, invasion, migration, and apoptosis were detected to explore the involvement of DPP10-AS1 and miR-127-3p in the colon cancer stem cell (CCSC) properties through gain- and loss-of function approaches. Furthermore, tumor xenograft in nude mice was conducted to investigate the effect of DPP10-AS1 and miR-127-3p on tumor growth in vivo . Poorly expressed DPP10-AS1 and ADCY1, while highly expressed miR-127-3p were found in CCSCs. Low expression of DPP10-AS1 was correlated with TNM stage, lymphatic node metastasis, and tumor differentiation. Upregulation of DPP10-AS1 increased ADCY1 protein expression, decreased the protein expression of CCSC-related factors, inhibited sphere formation, colony formation, proliferation, invasion and migration, and accelerated apoptosis of HT-29 and SW480 cells by suppressing the expression of miR-127-3p. Further, the above in vitro findings were also confirmed by in vivo assays. Taken together, this study demonstrates that DPP10-AS1 inhibits CCSC proliferation by regulating miR-127-3p and ADCY1, providing fresh insight into a promising novel treatment strategy for colon cancer.

Cyclic adenosine monophosphate (cAMP) is an important secondary messenger that controls carbon metabolism, type IVa pili biogenesis, and virulence in Pseudomonas aeruginosa . Precise manipulation of bacterial intracellular cAMP levels may enable tunable control of twitching motility or virulence, and optogenetic tools are attractive because they afford excellent spatiotemporal resolution and are easy to operate. Here, we developed an engineered P. aeruginosa strain (termed pactm ) with light-dependent intracellular cAMP levels through introducing a photoactivated adenylate cyclase gene ( bPAC ) into bacteria. On blue light illumination, pactm displayed a 15-fold increase in the expression of the cAMP responsive promoter and an 8-fold increase in its twitching activity. The skin lesion area of nude mouse in a subcutaneous infection model after 2-day pactm inoculation was increased 14-fold by blue light, making pactm suitable for applications in controllable bacterial host infection. In addition, we achieved directional twitching motility of pactm colonies through localized light illumination, which will facilitate the studies of contact-dependent interactions between microbial species.

Olfactory receptors (ORs) have diverse physiological roles in various cell types, beyond their function as odorant sensors in the olfactory epithelium. These previous findings have suggested that ORs could be diagnostic markers and promising therapeutic targets in several pathological conditions. In the current study, we sought to characterize the changes in the expression of ORs in the HaCaT human keratinocytes cell line exposed to ultraviolet (UV) light or inflammation, well-recognized stimulus for skin barrier disruption. We confirmed that major olfactory signaling components, including ORs, GNAL , Ric8b , and adenylate cyclase type 3, are highly expressed in HaCaT cells. We have also demonstrated that the 12 ectopic ORs detectable in HaCaT cells are more highly expressed in UV-irradiated or inflamed conditions than in normal conditions. We further assessed the specific OR-mediated biological responses of HaCaT cells in the presence of known odorant ligands of ORs and observed that specific ligand-activated ORs downregulate skin barrier genes in HaCaT cells. This study shows the potential of OR as a marker for skin barrier abnormalities. Further research is needed to explore how OR is implicated in the development and progression of barrier dysfunction.

Skeletal and glycemic traits have shared etiology, but the underlying genetic factors remain largely unknown. To identify genetic loci that may have pleiotropic effects, we studied Genome-wide association studies (GWASs) for bone mineral density and glycemic traits and identified a bivariate risk locus at 3q21. Using sequence and epigenetic modeling, we prioritized an adenylate cyclase 5 (ADCY5) intronic causal variant, rs56371916. This SNP changes the binding affinity of SREBP1 and leads to differential ADCY5 gene expression, altering the chromatin landscape from poised to repressed. These alterations result in bone- and type 2 diabetes-relevant cell-autonomous changes in lipid metabolism in osteoblasts and adipocytes. We validated our findings by directly manipulating the regulator SREBP1, the target gene ADCY5, and the variant rs56371916, which together imply a novel link between fatty acid oxidation and osteoblast differentiation. Our work, by systematic functional dissection of pleiotropic GWAS loci, represents a framework to uncover biological mechanisms affecting pleiotropic traits., Competing Interests: Declaration of interests E.S.L. serves on the Board of Directors for Codiak BioSciences and serves on the Scientific Advisory Board of F-Prime Capital Partners and Third Rock Ventures; he is also affiliated with several non-profit organizations including serving on the Board of Directors of the Innocence Project, Count Me In, and Biden Cancer Initiative and the Board of Trustees for the Parker Institute for Cancer Immunotherapy. He has served and continues to serve on various federal advisory committees. C.M.L. received research support from Bayer Ag and Novo Nordisk and received honoraria or consultancy fees from Pfizer. D.P.K. serves as a member of a scientific advisory committee for Solarea Bio, has received grants to his institution from Radius Health and Amgen, and receives royalties for publication in UpToDate., (Published by Elsevier Inc.)

Incretin hormones analogues, including glucagon-like peptide type 1 (GLP-1), exhibit complex glucose-lowering, anorexigenic, and cardioprotective properties. Mechanisms of action of GLP-1 and its analogues are well known for pancreatic β-cells, hepatocytes, and other tissues. Nevertheless, local effects of GLP-1 and its analogues in adipose tissue remain unclear. In the present work effects of the GLP-1 synthetic analogue, liraglutide, on adipogenesis and insulin sensitivity of the 3T3-L1 adipocytes were examined. Enhancement of insulin sensitivity of mature adipocytes by the GLP-1 synthetic analogue liraglutide mediated by adenylate cyclase was demonstrated. The obtained results imply existence of the positive direct insulin-sensitizing effect of liraglutide on mature adipocytes.

Vitiligo is autoimmune, acquired, idiopathic, chronic, and progressive de/hypopigmentary cutaneous condition that targets the cell-producing pigment called melanin. It binds to a thread of great disappointment and emotional stress in societies. Combining multiple stress-related theories like toxic compound accumulation, autoimmunity, mutations, altered cellular environment, infection, impaired migration/proliferation, and immunological mismatch of anti-melanocyte and self-reactive T-cells that cause melanocytes damage is formulated resulting in vitiligo. Vitiligo has an orphan status for drug synthesis. Still, different therapies are available, with topical steroids and narrow-band ultraviolet-B monotherapy being the most common treatments, others including medical, physical, or surgical, but not effective. Each modality has its baggage of disadvantages and side effects. Stimulation of the transcriptional process for melanogenesis is mainly achieved by the cAMP-dependent activation of several melanogenic genes by MITF. In this review, we summarized that cAMP encourages the expression of the enzyme tyrosinase, TYRP1, TYRP2, and most other biological effects of cAMP are mediated through the cAMP-dependent PKA pathway resulting in CREB phosphorylation. It has been shown that TYRP1 and 2 do not have cAMP response elements (CREs) in promoting regions; the regulation of these genes by cAMP occurs through the direct participation of MITF during melanogenesis. The available medicines, therefore, only provide symptomatic relief, but do not stop the disease progression. In addition, the treatment process needs to be changed; existing approaches need to be overlooked for patients who are suffering and therefore analyze its efficacy and safety to achieve a favorable risk-benefit ratio.

Hydroxycarboxylic acid receptor 2 (HCA 2 ) is vital for sensing intermediates of metabolism, including β-hydroxybutyrate and butyrate. It also regulates profound anti-inflammatory effects in various tissues, indicating that HCA 2 may serve as an essential therapeutic target for mediating inflammation-associated diseases. Butyrate and niacin, endogenous and exogenous ligands of HCA 2 , have been reported to play an essential role in maintaining intestinal homeostasis. HCA 2 , predominantly expressed in diverse immune cells, is also present in intestinal epithelial cells (IECs), where it regulates the intricate communication network between diet, microbiota, and immune cells. This review summarizes the physiological role of HCA 2 in intestinal homeostasis and its pathological role in intestinal inflammation and cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li, McCafferty and Judd.)

Fibroblast growth factor 9 (FGF9) has long been assumed to modulate multiple biological processes, yet very little is known about the impact of FGF9 on neurodevelopment. Herein, we found that loss of Fgf9 in olig1 progenitor cells induced epilepsy in mice, with pathological changes in the cortex. Then depleting Fgf9 in different neural populations revealed that epilepsy was associated with GABAergic neurons. Fgf9 CKO in GABAergic neuron (CKO VGAT ) mice exhibited not only the most severe seizures, but also the most severe growth retardation and highest mortality. Fgf9 deletion in CKO VGAT mice caused neuronal apoptosis and decreased GABA expression, leading to a GABA/Glu imbalance and epilepsy. The adenylate cyclase/cyclic AMP and ERK signaling pathways were activated in this process. Recombinant FGF9 proteoliposomes could significantly decrease the number of seizures. Furthermore, the decrease of FGF9 was commonly observed in serum of epileptic patients, especially those with focal seizures. Thus, FGF9 plays essential roles in GABAergic neuron survival and epilepsy pathology, which could serve as a new target for the treatment of epilepsy.

Cognitive dysfunction and mood changes are prevalent and especially taxing issues for patients with systemic lupus erythematosus (SLE). Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and its cognate receptor Fn14 have been shown to play an important role in neurocognitive dysfunction in murine lupus. We profiled and compared gene expression in the cortices of MRL/+, MRL/ lpr (that manifest lupus-like phenotype) and MRL/ lpr -Fn14 knockout (Fn14ko) adult female mice to determine the transcriptomic impact of TWEAK/Fn14 on cortical gene expression in lupus. We found that the TWEAK/Fn14 pathway strongly affects the expression level, variability and coordination of the genomic fabrics responsible for neurotransmission and chemokine signaling. Dysregulation of the Phosphoinositide 3-kinase (PI3K)-AKT pathway in the MRL/ lpr lupus strain compared with the MRL/+ control and Fn14ko mice was particularly prominent and, therefore, promising as a potential therapeutic target, although the complexity of the transcriptomic fabric highlights important considerations in in vivo experimental models.

The aim of the present study was to investigate the effects of microRNA (miR)‑142‑3p on neuropathic pain caused by sciatic nerve injury in chronic compression injury (CCI) rats, and further investigate its mechanism. Rat experiments were divided into four parts in the study. In the first part, the rats were divided into the Sham and CCI groups. The expression of miR‑142‑3p, AC9 and cAMP were detected. In the second part, the rats were divided into the Sham, CCI, miR‑142‑3p mimic, mimic‑negative control (NC), miR‑142‑3p small interfering RNA (siRNA) and siRNA‑NC groups. The expression of cAMP and the levels of AMPK pathway‑related proteins were detected. In the third part, the rats were randomly divided into Sham, CCI, AC9 mimic, mi‑NC, AC9 siRNA and si‑NC groups. Double luciferase reporter assay was used to analyse the targeting relationship between miR‑142‑3p and AC9. In the fourth part, the rats were divided into the Sham, CCI, miR‑142‑3p siRNA, AC9 mimic, miR‑142‑3p siRNA + AC9 siRNA, cAMP activator (Forskolin) and miR‑142‑3p siRNA + cAMP inhibitor groups. The expression of miR‑142‑3p was significantly increased while AC9 and cAMP expression significantly decreased in CCI rats. However, AC9 overexpression significantly increased the levels of cAMP protein. Luciferase reporter assay also proved that AC9 is the target gene of miR‑142‑3p. Moreover, miR‑142‑3p silencing was found to reduce neuropathic pain in CCI rats by upregulating the expression of AC9. It was also found that cAMP activation can relieve neuropathic pain and promote the expression of AMPK‑related proteins in CCI rats. Silencing miR‑142‑3p can target AC9 to reduce the expression of inflammatory factors and neuropathic pain in CCI rats by increasing the expression of cAMP/AMPK pathway‑related proteins.

Astrocytes play a key role in brain homeostasis and functions such as memory. Specifically, astrocytes express multiple receptors that transduce signals via the second messenger cAMP. However, the involvement of astrocytic cAMP in animal behavior and the underlying glial-neuronal interactions remains largely unknown. Here, we show that an increase in astrocytic cAMP is sufficient to induce synaptic plasticity and modulate memory. We developed a method to increase astrocytic cAMP levels in vivo using photoactivated adenylyl cyclase and found that increased cAMP in hippocampal astrocytes at different time points facilitated memory formation but interrupted memory retention via NMDA receptor-dependent plasticity. Furthermore, we found that the cAMP-induced modulation of memory was mediated by the astrocyte-neuron lactate shuttle. Thus, our study unveils a role of astrocytic cAMP in brain function by providing a tool to modulate astrocytic cAMP in vivo., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Multiple sclerosis is an idiopathic and autoimmune associated motor neuron disorder that affects myelinated neurons in specific brain regions of young people, especially females. MS is characterized by oligodendrocytes destruction further responsible for demyelination, neuroinflammation, mitochondrial abnormalities, oxidative stress and neurotransmitter deficits associated with motor and cognitive dysfunctions, vertigo and muscle weakness. The limited intervention of pharmacologically active compounds like interferon-β, mitoxantrone, fingolimod and monoclonal antibodies used clinically are majorly associated with adverse drug reactions. Pre-clinically, gliotoxin ethidium bromide mimics the behavioral and neurochemical alterations in multiple sclerosis- like in experimental animals associated with the down-regulation of adenyl cyclase/cAMP/CREB, which is further responsible for a variety of neuropathogenic factors. Despite the considerable investigation of neuroprotection in curing multiple sclerosis, some complications still remain. The available medications only provide symptomatic relief but do not stop the disease progression. In this way, the development of unused beneficial methods tends to be ignored. The limitations of the current steady treatment may be because of their activity at one of the many neurotransmitters included or their failure to up direct signaling flag bearers detailed to have a vital part in neuronal sensitivity, biosynthesis of neurotransmitters and its discharge, development, and separation of the neuron, synaptic versatility and cognitive working. Therefore, the current review strictly focused on the exploration of various clinical and pre-clinical features available for multiple sclerosis to understand the pathogenic mechanisms and to introduce pharmacological interventions associated with the upregulation of intracellular adenyl cyclase/cAMP/CREB activation to ameliorate multiple sclerosis-like features., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Haematopoietic stem cells (HSCs) reside in specialized microenvironments in the bone marrow-often referred to as 'niches'-that represent complex regulatory milieux influenced by multiple cellular constituents, including nerves 1,2 . Although sympathetic nerves are known to regulate the HSC niche 3-6 , the contribution of nociceptive neurons in the bone marrow remains unclear. Here we show that nociceptive nerves are required for enforced HSC mobilization and that they collaborate with sympathetic nerves to maintain HSCs in the bone marrow. Nociceptor neurons drive granulocyte colony-stimulating factor (G-CSF)-induced HSC mobilization via the secretion of calcitonin gene-related peptide (CGRP). Unlike sympathetic nerves, which regulate HSCs indirectly via the niche 3,4,6 , CGRP acts directly on HSCs via receptor activity modifying protein 1 (RAMP1) and the calcitonin receptor-like receptor (CALCRL) to promote egress by activating the Gα s /adenylyl cyclase/cAMP pathway. The ingestion of food containing capsaicin-a natural component of chili peppers that can trigger the activation of nociceptive neurons-significantly enhanced HSC mobilization in mice. Targeting the nociceptive nervous system could therefore represent a strategy to improve the yield of HSCs for stem cell-based therapeutic agents.

Quantitative measurement of receptor signaling by different ligands is important for understanding the mechanism of drug action and screening of drugs. Here, we describe a simple and cost-effective method of measuring adenylyl cyclase inhibition, one of the hallmarks of opioid receptor activation. The assay is based on bioluminescence resonance energy transfer (BRET) that involves transfection of a biosensor in human embryonic kidney (HEK)-293 cells stably transfected with μ-opioid receptor (μ receptor), enabling real-time measurement of cAMP levels.

Proteins interacting with G protein-coupled receptors (GPCRs) can modulate signal transduction of these receptors. However, the regulatory mechanisms of the interacting proteins are diverse and largely unknown. We have previously shown that Tctex-1 (or DYNLT1) can interact with the parathyroid hormone receptor (PTHR). In the present study, we investigated the role of Tctex-1 in the PTHR signaling and found that Tctex-1 augmented the PTHR-mediated G s /adenylyl cyclase (AC) pathway by activating AC regardless of the binding to PTHR. Furthermore, Tctex-1 directly bound to AC type 6. These data demonstrate a novel mechanism underlying GPCR/G s signaling regulated by Tctex-1., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Cyclic AMP and Ca 2+ are the first second or intracellular messengers identified, unveiling the cellular mechanisms activated by a plethora of extracellular signals, including hormones. Cyclic AMP generation is catalyzed by adenylyl cyclases (ACs), which convert ATP into cAMP and pyrophosphate. By the way, Ca 2+ , as energy, can neither be created nor be destroyed; Ca 2+ can only be transported, from one compartment to another, or chelated by a variety of Ca 2+ -binding molecules. The fine regulation of cytosolic concentrations of cAMP and free Ca 2+ is crucial in cell function and there is an intimate cross-talk between both messengers to fine-tune the cellular responses. Cancer is a multifactorial disease resulting from a combination of genetic and environmental factors. Frequent cases of cAMP and/or Ca 2+ homeostasis remodeling have been described in cancer cells. In those tumoral cells, cAMP and Ca 2+ signaling plays a crucial role in the development of hallmarks of cancer, including enhanced proliferation and migration, invasion, apoptosis resistance, or angiogenesis. This review summarizes the cross-talk between the ACs/cAMP and Ca 2+ intracellular pathways with special attention to the functional and reciprocal regulation between Orai1 and AC8 in normal and cancer cells.

Background: The ciliary beat of the airway epithelium, including the sinonasal epithelium, has a significant role in frontline defense and is thought to be controlled by the level of intracellular Ca2+. Involvement of calmodulin and adenylate/guanylate cyclases in the regulation of ciliary beats has been reported, and here we investigated the interrelation between these components of the ciliary beat regulatory pathway., Methods: The inferior turbinates were collected from 29 patients with chronic hypertrophic rhinitis/rhinosinusitis during endoscopic sinonasal surgery. The turbinate mucosa was cut into thin strips, and mucociliary movement was observed under a phase-contrast light microscope equipped with a high-speed digital video camera., Results: The ciliary beat frequency (CBF) was significantly increased by stimulation with 100 μM CALP3 (calmodulin agonist), which was completely suppressed by adding 100 µM SQ22536 (adenylate cyclase inhibitor) and 10 µM ODQ (guanylate cyclase inhibitor) together and by adding 1 µM KT5720 (protein kinase A inhibitor) and 1 µM KT5823 (protein kinase G inhibitor) together. The CBF was significantly increased by stimulation with 10 µM forskolin (adenylate cyclase activator) and 10 µM BAY41-2272 (guanylate cyclase activator) and by stimulation with 100 µM 8-bromo-cAMP (cAMP analog) and 100 µM 8-bromo-cGMP (cGMP analog), which was not changed by adding 1 µM calmidazolium (calmodulin antagonist)., Conclusions: These results confirmed that the regulatory pathway of ciliary beats in the human nasal mucosa involves calmodulin, adenylate/guanylate cyclases, and protein kinases A/G and indicate that adenylate/guanylate cyclases and protein kinases A/G act downstream of calmodulin, but not vice versa, and that these cyclases relay calmodulin signaling., (© 2021 S. Karger AG, Basel.)

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.

Cortical long-term potentiation (LTP) serves as a cellular model for chronic pain. As an important subtype of adenylyl cyclases (ACs), adenylyl cyclase subtype 1 (AC1) is critical for the induction of cortical LTP in the anterior cingulate cortex (ACC). Genetic deletion of AC1 or pharmacological inhibition of AC1 blocked behavioral allodynia in animal models of neuropathic and inflammatory pain. Our previous experiments have identified a lead candidate AC1 inhibitor, NB001, which is highly selective for AC1 over other AC isoforms, and found that NB001 is effective in inhibiting behavioral allodynia in animal models of chronic neuropathic and inflammatory pain. However, previous experiments were carried out in adult male animals. Considering the potential gender difference as an important issue in researches of pain and analgesia, we investigated the effect of NB001 in female chronic pain animal models. We found that NB001, when administered orally, has an analgesic effect in female animal models of neuropathic and inflammatory pain without any observable side effect. Genetic deletion of AC1 also reduced allodynia responses in models of neuropathic pain and chronic inflammation pain in adult female mice. In brain slices of adult female mice, bath application of NB001(20 μM) blocked the induction of LTP in ACC. Our results indicate that calcium-stimulated AC1 is required for injury-related cortical LTP and behavioral allodynia in both sexes of adult animals, and NB001 can be used as a potential therapeutic drug for treating neuropathic and inflammatory pain in man and woman.

The second messenger cyclic adenosine monophosphate (cAMP) regulates numerous functions in both benign melanocytes and melanoma cells. cAMP is generated from two distinct sources, transmembrane and soluble adenylyl cyclases (tmAC and sAC, respectively), and is degraded by a family of proteins called phosphodiesterases (PDEs). cAMP signaling can be regulated in many different ways and can lead to varied effects in melanocytes. It was recently revealed that distinct cAMP signaling pathways regulate pigmentation by either altering pigment gene expression or the pH of melanosomes. In the context of melanoma, many studies report seemingly contradictory roles for cAMP in tumorigenesis. For example, cAMP signaling has been implicated in both cancer promotion and suppression, as well as both therapy resistance and sensitization. This conundrum in the field may be explained by the fact that cAMP signals in discrete microdomains and each microdomain can mediate differential cellular functions. Here, we review the role of cAMP signaling microdomains in benign melanocyte biology, focusing on pigmentation, and in melanomagenesis., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Photoactivated adenylyl cyclase (PAC) was first discovered to be a sensor for photoavoidance in the flagellate Euglena gracilis. PAC is a flavoprotein that catalyzes the production of cAMP upon illumination with blue light, which enables us to optogenetically manipulate intracellular cAMP levels in various biological systems. Recent progress in genome sequencing has revealed several related proteins in bacteria and ameboflagellates. Among them, the PACs from sulfur bacterium Beggiatoa sp. and cyanobacterium Oscillatoria acuminata have been well characterized, including their crystalline structure. Although there have not been many reported optogenetic applications of PACs so far, they have the potential to be used in various fields within bioscience.

Inositol trisphosphate (IP 3 ) is a Ca 2+ -mobilizing second messenger shown to modulate atrial muscle contraction and is thought to contribute to atrial fibrillation. Cellular pathways underlying IP 3 actions in cardiac tissue remain poorly understood, and the work presented here addresses the question whether IP 3 -mediated Ca 2+ release from the sarcoplasmic reticulum is linked to adenylyl cyclase activity including Ca 2+ -stimulated adenylyl cyclases (AC1 and AC8) that are selectively expressed in atria and sinoatrial node (SAN). Immunocytochemistry in guinea pig atrial myocytes identified colocalization of type 2 IP 3 receptors with AC8, while AC1 was located in close vicinity. Intracellular photorelease of IP 3 by UV light significantly enhanced the amplitude of the Ca 2+ transient (CaT) evoked by electrical stimulation of atrial myocytes (31 ± 6% increase 60 s after photorelease, n = 16). The increase in CaT amplitude was abolished by inhibitors of adenylyl cyclases (MDL-12,330) or protein kinase A (H89), showing that cAMP signaling is required for this effect of photoreleased IP 3 . In mouse, spontaneously beating right atrial preparations, phenylephrine, an α-adrenoceptor agonist with effects that depend on IP 3 -mediated Ca 2+ release, increased the maximum beating rate by 14.7 ± 0.5%, n = 10. This effect was substantially reduced by 2.5 µmol/L 2-aminoethyl diphenylborinate and abolished by a low dose of MDL-12,330, observations which are again consistent with a functional interaction between IP 3 and cAMP signaling involving Ca 2+ stimulation of adenylyl cyclases in the SAN pacemaker. Understanding the interaction between IP 3 receptor pathways and Ca 2+ -stimulated adenylyl cyclases provides important insights concerning acute mechanisms for initiation of atrial arrhythmias. NEW & NOTEWORTHY This study provides evidence supporting the proposal that IP 3 signaling in cardiac atria and sinoatrial node involves stimulation of Ca 2+ -activated adenylyl cyclases (AC1 and AC8) by IP 3 -evoked Ca 2+ release from junctional sarcoplasmic reticulum. AC8 and IP 3 receptors are shown to be located close together, while AC1 is nearby. Greater understanding of these novel aspects of the IP 3 signal transduction mechanism is important for future study in atrial physiology and pathophysiology, particularly atrial fibrillation.

Genetically encoded fluorescent biosensors are powerful tools for studying complex signaling in the nervous system, and now both Ca2+ and voltage sensors are available to study the signaling behavior of entire neural circuits. There is a pressing need for improved sensors, but improving them is challenging because testing them involves a low throughput, labor-intensive processes. Our goal was to create synthetic, excitable cells that can be activated with brief pulses of blue light and serve as a medium throughput platform for screening the next generation of sensors. In this live cell system, blue light activates an adenylyl cyclase enzyme (bPAC) that increases intracellular cAMP (Stierl M et al. 2011). In turn, the cAMP opens a cAMP-gated ion channel. This produces slow, whole-cell Ca2+ transients and voltage changes. To increase the speed of these transients, we add the inwardly rectifying potassium channel Kir2.1, the bacterial voltage-gated sodium channel NAVROSD, and Connexin-43. The result is a highly reproducible, medium-throughput, live cell system that can be used to screen voltage and Ca2+ sensors., Competing Interests: The authors have read the journal’s policy and have the following potential competing interests. Thomas Hughes works for both Montana State University and Montana Molecular. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents associated with this work to declare. All of the materials are available through Addgene in plasmid form with no restrictions. Montana Molecular does market the individual components packaged in BacMam virus for ease of use

The catecholamines norepinephrine and epinephrine are important regulators of vertebrate physiology. Insects such as honeybees do not synthesize these neuroactive substances. Instead, they use the phenolamines tyramine and octopamine for similar physiological functions. These biogenic amines activate specific members of the large protein family of G protein-coupled receptors (GPCRs). Based on molecular and pharmacological data, insect octopamine receptors were classified as either α- or β-adrenergic-like octopamine receptors. Currently, one α- and four β-receptors have been molecularly and pharmacologically characterized in the honeybee. Recently, an α 2 -adrenergic-like octopamine receptor was identified in Drosophila melanogaster (DmOctα2R). This receptor is activated by octopamine and other biogenic amines and causes a decrease in intracellular cAMP ([cAMP] i ). Here, we show that the orthologous receptor of the honeybee (AmOctα2R), phylogenetically groups in a clade closely related to human α 2 -adrenergic receptors. When heterologously expressed in an eukaryotic cell line, AmOctα2R causes a decrease in [cAMP] i . The receptor displays a pronounced preference for octopamine over tyramine. In contrast to DmOctα2R, the honeybee receptor is not activated by serotonin. Its activity can be blocked efficiently by 5-carboxamidotryptamine and phentolamine. The functional characterization of AmOctα2R now adds a sixth member to this subfamily of monoaminergic receptors in the honeybee and is an important step towards understanding the actions of octopamine in honeybee behavior and physiology.

In spite of the positive effects of bacteria on health, certain species are harmful, and therefore, animals must weigh nutritional benefits against negative post-ingestion consequences and adapt their behavior accordingly. Here, we use Drosophila to unravel how the immune system communicates with the brain, enabling avoidance of harmful foods. Using two different known fly pathogens, mildly pathogenic Erwinia carotovora (Ecc15) and highly virulent Pseudomonas entomophila (Pe), we analyzed preference behavior in naive flies and after ingestion of either of these pathogens. Although survival assays confirmed the harmful effect of pathogen ingestion, naive flies preferred the odor of either pathogen to air and also to harmless mutant bacteria, suggesting that flies are not innately repelled by these microbes. By contrast, feeding assays showed that, when given a choice between pathogenic and harmless bacteria, flies-after an initial period of indifference-shifted to a preference for the harmless strain, a behavior that lasted for several hours. Flies lacking synaptic output of the mushroom body (MB), the fly's brain center for associative memory formation, lost the ability to distinguish between pathogenic and harmless bacteria, suggesting this to be an adaptive behavior. Interestingly, this behavior relied on the immune receptors PGRP-LC and -LE and their presence in octopaminergic neurons. We postulate a model wherein pathogen ingestion triggers PGRP signaling in octopaminergic neurons, which in turn relay the information about the harmful food source directly or indirectly to the MB, where an appropriate behavioral output is generated., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Since their discovery in the mid-1990s, regulator of G protein signaling (RGS) proteins have emerged as key regulators of signaling through G protein-coupled receptors. Among the over 20 known RGS proteins, RGS2 has received increasing interest as a potential therapeutic drug target with broad clinical implications. RGS2 is a member of the R4 subfamily of RGS proteins and is unique in that it is selective for G α q Despite only having an RGS domain, responsible for the canonical GTPase activating protein activity, RGS2 can regulate additional processes, such as protein synthesis and adenylate cyclase activity, through protein-protein interactions. Here we provide an update of the current knowledge of RGS2 function as it relates to molecular mechanisms of regulation as well as its potential role in regulating a number of physiologic systems and pathologies, including cardiovascular disease and central nervous system disorders, as well as various forms of cancer. SIGNIFICANCE STATEMENT: Regulator of G protein signaling (RGS) proteins represent an exciting class of novel drug targets. RGS2, in particular, could have broad clinical importance. As more details are emerging on the regulation of RGS2 in various physiological systems, the potential utility of this small protein in therapeutic development is increasing., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: In this study, we investigated the effect of forskolin (FSK, a selective adenylate cyclase agonist) on the automatic diastolic depolarization of sinus node cells (SNC) with hypoxia/reoxygenation (H/R) injury., Methods: The SNC of the newborn rat was randomly assigned into the control group, the H/R (H/R injury) group, or the H/R + FSK (H/R injury + FSK treatment) group. Patch-clamp was performed to record the action potential and electrophysiological changes. The cellular distribution of intracellular calcium concentration was analyzed by fluorescence staining., Results: Compared with the control cells, spontaneous pulsation frequency (SPF) and diastolic depolarization rate (DDR) of H/R cells were reduced from 244.3 ± 10.6 times/min and 108.7 ± 7.8 mV/s to 130.5 ± 7.6 times/min and 53.4 ± 6.5 mV/s, respectively. FSK significantly increased SPF and DDR of H/R cells to 208.3 ± 8.3 times/min and 93.2 ± 8.9 mV/s (n = 15, both p < 0.01), respectively. H/R reduced the current densities of I f , I Ca,T and inward I NCX , which were significantly increased by 10 μM FSK treatment (n = 15, p < 0.01). Furthermore, reduced expression of HCN4 and NCX1.1 channel protein were significantly increased by FSK. Inhibitor studies showed that both SQ22536 (a selective adenylate cyclase inhibitor) and H89 (a selective protein kinases A [PKA] inhibitor) blocked the effects of FSK on SPF and DDR., Conclusions: H/R causes pacemaker dysfunction in newborn rat sinoatrial node cells leading to divergence of the DD and the slow of spontaneous APs, which change can be dramatically reversed by FSK through increasing I NCX and I f current in H/R injury.

Bacterial photoactivated adenylyl cyclase (bPAC) has been widely used in signal transduction research. However, due to its low two-photon absorption, bPAC cannot be efficiently activated by two-photon (2P) excitation. Taking advantage of the high two-photon absorption of monomeric teal fluorescent protein 1 (mTFP1), we herein developed 2P-activatable bPAC (2pabPAC), a fusion protein consisting of bPAC and mTFP1. In 2pabPAC, the energy absorbed by mTFP1 excites bPAC by Fürster resonance energy transfer (FRET) at ca . 43% efficiency. The light-induced increase in cAMP was monitored by a red-shifted FRET biosensor for PKA. In 3D MDCK cells and mouse liver, PKA was activated at single-cell resolution under a 2P microscope. We found that PKA activation in a single hepatocyte caused PKA activation in neighboring cells, indicating the propagation of PKA activation. Thus, 2pabPAC will provide a versatile platform for controlling the cAMP signaling pathway and investigating cell-to-cell communication in vivo .

The recent discovery of sensory (tastant and odorant) G protein-coupled receptors on the smooth muscle of human bronchi suggests unappreciated therapeutic targets in the management of obstructive lung diseases. Here we have characterized the effects of a wide range of volatile odorants on the contractile state of airway smooth muscle (ASM) and uncovered a complex mechanism of odorant-evoked signaling properties that regulate excitation-contraction (E-C) coupling in human ASM cells. Initial studies established multiple odorous molecules capable of increasing intracellular calcium ([Ca 2+ ] i ) in ASM cells, some of which were (paradoxically) associated with ASM relaxation. Subsequent studies showed a terpenoid molecule (nerol)-stimulated OR2W3 caused increases in [Ca 2+ ] i and relaxation of ASM cells. Of note, OR2W3-evoked [Ca 2+ ] i mobilization and ASM relaxation required Ca 2+ flux through the store-operated calcium entry (SOCE) pathway and accompanied plasma membrane depolarization. This chemosensory odorant receptor response was not mediated by adenylyl cyclase (AC)/cyclic nucleotide-gated (CNG) channels or by protein kinase A (PKA) activity. Instead, ASM olfactory responses to the monoterpene nerol were predominated by the activity of Ca 2+ -activated chloride channels (TMEM16A), including the cystic fibrosis transmembrane conductance regulator (CFTR) expressed on endo(sarco)plasmic reticulum. These findings demonstrate compartmentalization of Ca 2+ signals dictates the odorant receptor OR2W3-induced ASM relaxation and identify a previously unrecognized E-C coupling mechanism that could be exploited in the development of therapeutics to treat obstructive lung diseases., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Motile cilia are hairlike structures that line the respiratory and reproductive tracts and the middle ear and generate fluid flow in these organs via synchronized beating. Cilium growth is a highly regulated process that is assumed to be important for flow generation. Recently, Kif19a, a kinesin residing at the cilia tip, was identified to be essential for ciliary length control through its microtubule depolymerization function. However, there is a lack of information on the nature of proteins and the integrated signaling mechanism regulating growth of motile cilia. Here, we report that adenylate cyclase 6 (AC6), a highly abundant AC isoform in airway epithelial cells, inhibits degradation of Kif19a by inhibiting autophagy, a cellular recycling mechanism for damaged proteins and organelles. Using epithelium-specific knockout mice of AC6, we demonstrated that AC6 knockout airway epithelial cells have longer cilia compared with the WT cells because of decreased Kif19a protein levels in the cilia. We demonstrated in vitro that AC6 inhibits AMP-activated kinase (AMPK), an important modulator of cellular energy-conserving mechanisms, and uncouples its binding with ciliary kinesin Kif19a. In the absence of AC6, activation of AMPK mobilizes Kif19a into autophagosomes for degradation in airway epithelial cells. Lower Kif19a levels upon pharmacological activation of AMPK in airway epithelial cells correlated with elongated cilia and vice versa. In all, the AC6-AMPK pathway, which is tunable to cellular cues, could potentially serve as one of the crucial ciliary growth checkpoints and could be channeled to develop therapeutic interventions for cilia-associated disorders., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Arora et al.)

Nesfatin-1 (NESF) and NESF-like peptide (NLP), encoded in nucleobindin 2 and 1 (NUCB2 and NUCB1), respectively, are orphan ligands and metabolic factors. We hypothesized that NESF and NLP suppress growth hormone (GH) synthesis, and aimed to determine whether mammalian somatotrophs are a source and site of action of these peptides. Using immortalized rat somatotrophs (GH3 cells), NUCB expression was determined by qPCR, immunofluorescence and Western blot. NESF and NLP binding to GH3 cells was tested using fluorescence imaging. Both time- and concentration-dependent studies were performed to test whether NESF and NLP affect GH. Moreover, the ability of these peptides to modulate the effects of ghrelin, and cell-signaling pathways were studied. GH3 cells express NUCB mRNAs and protein. Labeled NESF and NLP bind to the surface of GH3 cells, and incubation with either NESF or NLP decreased GH mRNA and protein expression, downregulated pit-1 mRNA, and blocked the GH stimulatory effects of ghrelin. Pre-incubation with either of these peptides reduced CREB phosphorylation by an AC-activator, but not when PKA was directly activated by a cAMP analog. Our results indicate that rat somatotrophs are a source of NUCBs, and that NESF and NLP downregulate GH synthesis through the AC/PKA/CREB signaling pathway.

Aims: To investigate the differentially expressed genes (DEGs) and molecular interaction in unstable atherosclerotic carotid plaques., Methods: Gene expression datasets GSE41571, GSE118481, and E-MTAB-2055 were analyzed. Co-regulated DEGs in at least two datasets were analyzed with the enrichment of Gene Ontology Biological Process (GO-BP), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein-protein interaction (PPI) networks, interrelationships between miRNAs/transcriptional factors, and their target genes and drug-gene interactions. The expression of notable DEGs in human carotid artery plaques and plasma was further identified., Results: The GO-BP enrichment analysis revealed that genes associated with inflammatory response, and extracellular matrix organization were altered. The KEGG enrichment analysis revealed that upregulated DEGs were enriched in the tuberculous, lysosomal, and chemokine signaling pathways, whereas downregulated genes were enriched in the focal adhesion and PI3K/Akt signaling pathway. Collagen type I alpha 2 chain (COL1A2), adenylate cyclase 3 (ADCY3), C-X-C motif chemokine receptor 4 (CXCR4), and TYRO protein tyrosine kinase binding protein (TYROBP) might play crucial roles in the PPI networks. In drug-gene interactions, colonystimulating factor-1 receptor had the most drug interactions. Insulin-like growth factor binding protein 6 (IGFBP6) was markedly downregulated in unstable human carotid plaques and plasma. Under a receiver operating characteristic curve analysis, plasma IGFBP6 had a significant discriminatory power (AUC, 0.894; 95% CI, 0.810-0.977), with a cutoff value of 142.08 ng/mL., Conclusions: The genes COL1A2, ADCY3, CXCR4, and TYROBP are promising targets for the prevention of unstable carotid plaque formation. IGFBP6 may be an important biomarker for predicting vulnerable plaques.

Hepatic gluconeogenesis is the main pathway for blood glucose maintenance activated during fasting. Retardation of insulin action, such as in diabetes mellitus, activates gluconeogenesis during the fed state. While the role of progesterone (P4) in diabetes is controversial, the P4 receptor, progesterone receptor membrane component 1 (PGRMC1), is known to stimulate pancreatic insulin secretion. We investigated the role of P4, via hepatic PGRMC1, during gluconeogenesis. The PGRMC1 binding chemical, AG-205, induced PGRMC1 monomer (25 kDa) abundance, and increased PEPCK expression and glucose production in parallel with cyclic AMP (cAMP) induction in Hep3B cells. PGRMC1-mediated cyclic AMP was inhibited by an adenylate cyclase inhibitor (MDL-12,330A). PEPCK suppression in Pgrmc1 KO hepatocyte was not observed after treatment of MDL-12,330A. PGRMC1 knockdown or overexpression systems in Hep3B cells confirmed that PGRMC1 mediates PEPCK expression via phosphorylation of cAMP-response element binding protein (CREB). CREB phosphorylation and PEPCK expression in primary hepatocytes were greater than that in PGRMC1 knock-out hepatocytes. Progesterone increased PGRMC1 expression, which induced cAMP and PEPCK induction and glucose production. In vivo, P4 suppressed gluconeogenesis following plasma insulin induction under normal conditions in a mouse model. However, P4 increased blood glucose via gluconeogenesis in parallel with increases in PGRMC1 and PEPCK expression in mice in both insulin-deficient and insulin-resistant conditions. We conclude that P4 increases hepatic glucose production via PGRMC1, which may exacerbate hyperglycaemia in diabetes where insulin action is limited.

The mechanisms underlying subcellular targeting of cAMP-generating adenylyl cyclases and processes regulated by their compartmentalization are poorly understood. Here, we identify Ankmy2 as a repressor of the Hedgehog pathway via adenylyl cyclase targeting. Ankmy2 binds to multiple adenylyl cyclases, determining their maturation and trafficking to primary cilia. Mice lacking Ankmy2 are mid-embryonic lethal. Knockout embryos have increased Hedgehog signaling and completely open neural tubes showing co-expansion of all ventral neuroprogenitor markers, comparable to the loss of the Hedgehog receptor Patched1. Ventralization in Ankmy2 knockout is completely independent of the Hedgehog pathway transducer Smoothened. Instead, ventralization results from the reduced formation of Gli2 and Gli3 repressors and early depletion of adenylyl cyclase III in neuroepithelial cilia, implicating deficient pathway repression. Ventralization in Ankmy2 knockout requires both cilia and Gli2 activation. These findings indicate that cilia-dependent adenylyl cyclase signaling represses the Hedgehog pathway and promotes morphogenetic patterning., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Second messenger signaling controls a surprisingly diverse range of processes in several eukaryotic pathogens. Molecular machinery and pathways involving these messengers thus hold tremendous opportunities for therapeutic interventions. Relative to Ca 2+ signaling, the knowledge of a crucial second messenger cyclic AMP (cAMP) and its signaling pathway is very scant in the intestinal parasite Entamoeba histolytica . In the current study, mining the available genomic resources, we have for the first time identified the cAMP signal transduction pathway of E. histolytica . Three heptahelical proteins with variable G-protein-coupled receptor domains, heterotrimeric G-proteins (Gα, Gβ, and Gγ subunits), soluble adenylyl cyclase, cyclase-associated protein, and enzyme carbonic anhydrase were identified in its genome. We could also identify several putative candidate genes for cAMP downstream effectors such as protein kinase A, A-kinase anchoring proteins, and exchange protein directly activated by the cAMP pathway. Using specific inhibitors against key identified targets, we could observe changes in the intracellular cAMP levels as well as defect in the rate of phagocytosis of red blood cells by the parasite E. histolytica . We thus strongly believe that characterization of some of these unexplored crucial signaling determinants will provide a paradigm shift in understanding the pathogenicity of this organism., (Copyright © 2020 Agarwal, Rath, Anand and Gourinath.)

Adenylyl cyclases (ACs) have a crucial role in many signal transduction pathways, in particular in the intricate control of cyclic AMP (cAMP) generation from adenosine triphosphate (ATP). Using homology models developed from existing structural data and docking experiments, we have carried out all-atom, microsecond-scale molecular dynamics simulations on the AC5 isoform of adenylyl cyclase bound to the inhibitory G-protein subunit Gαi in the presence and in the absence of ATP. The results show that Gαi has significant effects on the structure and flexibility of adenylyl cyclase, as observed earlier for the binding of ATP and Gsα. New data on Gαi bound to the C1 domain of AC5 help explain how Gαi inhibits enzyme activity and obtain insight on its regulation. Simulations also suggest a crucial role of ATP in the regulation of the stimulation and inhibition of AC5.

We investigated the vasodilatory effects of empagliflozin (a sodium-glucose co-transporter 2 inhibitor) and the underlying mechanisms using rabbit aorta. Empagliflozin induced vasodilation in a concentration-dependent manner independently of the endothelium. Likewise, pretreatment with the nitric oxide synthase inhibitor L-NAME or the SKca inhibitor apamin together with the IKca inhibitor TRAM-34 did not impact the vasodilatory effects of empagliflozin. Pretreatment with the adenylyl cyclase inhibitor SQ22536 or a guanylyl cyclase inhibitor ODQ or a protein kinase A (PKA) inhibitor KT5720 also did not alter the vasodilatory response of empagliflozin. However, the vasodilatory effects of empagliflozin were significantly reduced by pretreatment with the protein kinase G (PKG) inhibitor KT5823. Although application of the ATP-sensitive K + (K ATP ) channel inhibitor glibenclamide, large-conductance Ca 2+ -activated K + (BK Ca ) channel inhibitor paxilline, or inwardly rectifying K + (Kir) channel inhibitor Ba 2+ did not impact the vasodilatory effects of empagliflozin, pretreatment with the voltage-dependent K + (Kv) channel inhibitor 4-AP reduced the vasodilatory effects of empagliflozin. Pretreatment with DPO-1 (Kv1.5 channel inhibitor), guangxitoxin (Kv2.1 channel inhibitor), or linopirdine (Kv7 channel inhibitor) had little effect on empagliflozin-induced vasodilation. Application of nifedipine (L-type Ca 2+ channel inhibitor) or thapsigargin (sarco-endoplasmic reticulum Ca 2+ -ATPase pump inhibitor) did not impact empagliflozin-induced vasodilation. Therefore, empagliflozin induces vasodilation by activating PKG and Kv channels., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Paramecium shows rapid forward swimming due to increased beat frequency of cilia in normal (forward swimming) direction in response to various kinds of stimuli applied to the cell surface that cause K + -outflow accompanied by a membrane hyperpolarization. Some adenylate cyclases are known to be functional K + channels in the membrane. Using gene-specific knockdown methods, we examined nine paralogues of adenylate cyclases in P. tetraurelia to ascertain whether and how they are involved in the mechanical stimulus-induced hyperpolarization-coupled acceleration of forward swimming. Results demonstrated that knockdown of the adenylate cyclase 1 (ac1)-gene and 2 (ac2)-gene inhibited the acceleration of forward swimming in response to mechanical stimulation of the cell, whereas that spared the acceleration response to external application of 8-Br-cAMP and dilution of extracellular [K + ] induced hyperpolarization. Electrophysiological examination of the knockdown cells revealed that the hyperpolarization-activated inward K + current is smaller than that of a normal cell. Our results suggest that AC1 and AC2 are involved in the mechanical stimulus-induced acceleration of ciliary beat in Paramecium., (© 2020 International Society of Protistologists.)

This study built an OA model in rats by monosodium iodoacetate (MIA) injection to determine the effects and mechanism of the voltage-dependent calcium channel subunit alpha-2/delta-1 (CACNA2D1)-calcitonin gene-related protein (CGRP) pathway in osteoarthritis (OA)-induced ongoing pain. CACNA2D1 expression was measured by qPCR assay, western blotting assay, and immunofluorescence. Pain behaviors in rats were assessed with the measurement of thermal paw withdrawal latency (PWL) and mechanical paw withdrawal threshold (PWT). The expression of CACNA2D1, neuropeptide Y (NPY), activating transcription factor 3 (ATF3), CGRP, protein kinase A (PKA), phosphorylated (p)-PKA, adenylyl cyclase (AC), protein kinase C (PKC), p-PKC, phospholipase C (PLC), and mitogen-activated protein kinase (MAPK) signaling pathway proteins were measured, OA rats had higher CACNA2D1 expression than normal rats. Knockdown of CACNA2D1 led to the elevation of the pain threshold of OA rats, and CACNA2D1 over-expression decreased the pain threshold of normal rats. Moreover, CACNA2D1 over-expression inhibited the expression of CGRP, up-regulated the expressions of NPY, ATF3, p-PKA, AC, p-PKC, PLC, p-Jun N-terminal kinase (JNK), and p-p38, and had no significant effect on phosphorylated extracellular signal-regulated kinase (p-ERK) expression in vivo and in vitro. Using this model of MIA-induced OA, we demonstrated that CACNA2D1 might be involved in the process of pain by modulating the CGRP and AC-PKA/PKC/MAPK signaling pathways in the dorsal root ganglion., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

A number of hormones and growth factors stimulate target cells via the second messenger pathways, which in turn regulate cellular phenotypes. Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that facilitates numerous signal transduction pathways; its production in cells is tightly balanced by ligand-stimulated receptors that activate adenylate cyclases (ACs), that is, "source" and by phosphodiesterases (PDEs) that hydrolyze it, that is, "sinks." Because it regulates various cellular functions, including cell growth and differentiation, gene transcription and protein expression, the cAMP signaling pathway has been exploited for the treatment of numerous human diseases. Reduction in cAMP is achieved by blocking "sources"; however, elevation in cAMP is achieved by either stimulating "source" or blocking "sinks." Here we discuss an alternative paradigm for the regulation of cellular cAMP via GIV/Girdin, the prototypical member of a family of modulators of trimeric GTPases, Guanine nucleotide Exchange Modulators (GEMs). Cells upregulate or downregulate cellular levels of GIV-GEM, which modulates cellular cAMP via spatiotemporal mechanisms distinct from the two most often targeted classes of cAMP modulators, "sources" and "sinks." A network-based compartmental model for the paradigm of GEM-facilitated cAMP signaling has recently revealed that GEMs such as GIV serve much like a "tunable valve" that cells may employ to finetune cellular levels of cAMP. Because dysregulated signaling via GIV and other GEMs has been implicated in multiple disease states, GEMs constitute a hitherto untapped class of targets that could be exploited for modulating aberrant cAMP signaling in disease states. This article is categorized under: Models of Systems Properties and Processes > Mechanistic Models Biological Mechanisms > Cell Signaling., (© 2020 Wiley Periodicals LLC.)

To test the hypothesis of STC-1 participation in maintenance of glucose homeostasis in fed and fasting (48 h) rats, we investigated that this hormone may be implicated in the regulation of renal gluconeogenesis pathway from lactate and lactate oxidation in renal cortex and medulla. Our results demonstrate the hSTC-1 role on lactate metabolism in the renal cortex and medulla from fed and fasting rats. hSTC-1 increased the gluconeogenesis activity in fed state in renal cortex, and this increase was induced by raise in Pck1 gene expression. In fasting animals hSTC-1 increase the renal medulla gluconeogenesis activity, but Pck1 gene expression was not alter. The stimulatory effect of hSTC-1 on 14 C-lactate oxidation occurred only in the renal cortex from fed rats. These findings show the hSTC-1 contribution to lactate homeostasis and supplies glucose to other tissues. This response may represent a strategy of action of STC-1 in response to fasting stress as postulated by different authors. On the other hand, hSTC-1 acts downstream of adenylcyclase pathway, decreasing the gluconeogenesis activity induced by cAMP intracellular increase or stimulating the phosphodiesterase activity in the renal cortex. However, no hSTC-1 effect on 14 C-lactate oxidation was found after increase in the intracellular cAMP. The findings also revealed that the renal cortex and medulla respond differently to hSTC-1, possibly due to the higher level of STC-1 gene expression in inner renal medulla than in renal cortex., Competing Interests: Declaration of competing interest We declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

An outbreak of the novel coronavirus (CoV) SARS-CoV-2, the causative agent of COVID-19 respiratory disease, infected millions of people since the end of 2019, led to high-level morbidity and mortality and caused worldwide social and economic disruption. There are currently no antiviral drugs available with proven efficacy or vaccines for its prevention. An understanding of the underlying cellular mechanisms involved in virus replication is essential for repurposing the existing drugs and/or the discovery of new ones. Endocytosis is the important mechanism of entry of CoVs into host cells. Endosomal maturation followed by the fusion with lysosomes are crucial events in endocytosis. Late endosomes and lysosomes are characterized by their acidic pH, which is generated by a proton transporter V-ATPase and required for virus entry via endocytic pathway. The cytoplasmic cAMP pool produced by soluble adenylyl cyclase (sAC) promotes V-ATPase recruitment to endosomes/lysosomes and thus their acidification. In this review, we discuss targeting the sAC-specific cAMP pool as a potential strategy to impair the endocytic entry of the SARS-CoV-2 into the host cell. Furthermore, we consider the potential impact of sAC inhibition on CoV-induced disease via modulation of autophagy and apoptosis.

The adenylyl cyclases (ACs) catalyze the production of the ubiquitous second messenger, cAMP, which in turns acts on a number of effectors and thus regulates a plethora of cellular functions. As the key enzymes in the highly evolutionarily conserved cAMP pathway, the ACs control the physiology of the cells, tissues, organs and organisms in health and disease. A comprehensive understanding of the specific role of the ACs in these processes of life requires a deep mechanistic understanding of structure and mechanisms of action of these enzymes. Here we highlight the exciting recent reports on the biochemistry and structure and higher order organization of the ACs and their signaling complexes. These studies have provided the glimpses into the principles of the AC-mediated homeostatic control of cellular physiology., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)