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The corticostriatal connection plays a crucial role in cognitive, emotional, and motor control. However, the specific roles and synaptic transmissions of corticostriatal connection are less studied, especially the corticostriatal transmission from the anterior cingulate cortex (ACC). Here, a direct glutamatergic excitatory synaptic transmission in the corticostriatal projection from the ACC is found. Kainate receptors (KAR)‐mediated synaptic transmission is increased in this corticostriatal connection both in vitro and in vivo seizure‐like activities. GluK1 containing KARs and downstream calcium‐stimulated adenylyl cyclase subtype 1 (AC1) are involved in the upregulation of KARs following seizure‐like activities. Inhibiting the activities of ACC or its corticostriatal connection significantly attenuated pentylenetetrazole (PTZ)‐induced seizure. Additionally, injection of GluK1 receptor antagonist UBP310 or the AC1 inhibitor NB001 both show antiepileptic effects. The studies provide direct evidence that KARs are involved in seizure activity in the corticostriatal connection and the KAR‐AC1 signaling pathway is a potential novel antiepileptic strategy. [ABSTRACT FROM AUTHOR]

Aim: To identify the physiological role of the acid‐base sensing enzyme, soluble adenylyl cyclase (sAC), in red blood cells (RBC) of the model teleost fish, rainbow trout. Methods: We used: (i) super‐resolution microscopy to determine the subcellular location of sAC protein; (ii) live‐cell imaging of RBC intracellular pH (pHi) with specific sAC inhibition (KH7 or LRE1) to determine its role in cellular acid‐base regulation; (iii) spectrophotometric measurements of haemoglobin–oxygen (Hb‐O2) binding in steady‐state conditions; and (iv) during simulated arterial‐venous transit, to determine the role of sAC in systemic O2 transport. Results: Distinct pools of sAC protein were detected in the RBC cytoplasm, at the plasma membrane and within the nucleus. Inhibition of sAC decreased the setpoint for RBC pHi regulation by ~0.25 pH units compared to controls, and slowed the rates of RBC pHi recovery after an acid‐base disturbance. RBC pHi recovery was entirely through the anion exchanger (AE) that was in part regulated by HCO3−‐dependent sAC signaling. Inhibition of sAC decreased Hb‐O2 affinity during a respiratory acidosis compared to controls and reduced the cooperativity of O2 binding. During in vitro simulations of arterial‐venous transit, sAC inhibition decreased the amount of O2 that is unloaded by ~11%. Conclusion: sAC represents a novel acid‐base sensor in the RBCs of rainbow trout, where it participates in the modulation of RBC pHi and blood O2 transport though the regulation of AE activity. If substantiated in other species, these findings may have broad implications for our understanding of cardiovascular physiology in vertebrates. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the effects and possible mechanisms of adenylate cyclase 1 (ADCY1) on pirarubicin‐induced cardiomyocyte injury. HL‐1 cells were treated with pirarubicin (THP) to induce intracellular toxicity, and the extent of damage to mouse cardiomyocytes was assessed using CCK‐8, Edu, flow cytometry, ROS, ELISA, RT‐qPCR and western blotting. THP treatment reduced the viability of HL‐1 cells, inhibited proliferation, induced apoptosis and triggered oxidative stress. In addition, the RT‐qPCR results revealed that ADCY1 expression was significantly elevated in HL‐1 cells, and molecular docking showed a direct interaction between ADCY1 and THP. Western blotting showed that ADCY1, phospho‐protein kinase A and GRIN2D expression were also significantly elevated. Knockdown of ADCY1 attenuated THP‐induced cardiotoxicity, possibly by regulating the ADCY1/PKA/GRIN2D pathway. [ABSTRACT FROM AUTHOR]

S‐Limonene (s‐Lim) is a monocyclic monoterpene found in a variety of plants and has been shown to present antioxidant and cardioprotective activity in experimental models of myocardial infarction. The aim of this study was to evaluate the potential mechanism by which s‐Lim exerts its antiarrhythmic effect, focusing on the blockade of β‐adrenoceptor (β‐AR) and its effects on various in vivo and in vitro parameters, including electrocardiogram (ECG) measurements, left ventricular developed pressure (LVDP), the β‐adrenergic pathway, sarcomeric shortening and L‐type calcium current (ICa,L). In isolated hearts, 10 μM of s‐Lim did not alter the ECG profile or LVPD. s‐Lim increased the heart rate corrected QT interval (QTc) (10.8%) at 50 μM and reduced heart rate at the concentrations of 30 (12.4%) and 50 μM (16.6%). s‐Lim (10 μM) also inhibited the adrenergic response evoked by isoproterenol (ISO) (1 μM) reducing the increased of heart rate, LVDP and ECG changes. In ventricular cardiomyocyte, s‐Lim antagonized the effect of dobutamine by preventing the increase of sarcomeric shortening, demonstrating a similar effect to atenolol (blocker β1‐AR). In vivo, s‐Lim antagonized the effect of ISO (agonists β1‐AR), presenting a similar effect to propranolol (a non‐selective blocker β‐AR). In ventricular cardiomyocyte, s‐Lim did not alter the voltage dependence for ICa,L activation or the ICa,L density. In addition, s‐Lim did not affect changes in the ECG effect mediated by 5 μM forskolin (an activator of adenylate cyclase). In an in vivo caffeine/ISO‐induced arrhythmia model, s‐Lim (1 mg/kg) presented antiarrhythmic action verified by a reduced arrhythmia score, heart rate, and occurrence of ventricular premature beats and inappropriate sinus tachycardia. These findings indicate that the antiarrhythmic activity of s‐Lim is related to blockade of β‐AR in the heart. [ABSTRACT FROM AUTHOR]

Bioelectronic medicine is emerging as a powerful approach for restoring lost endogenous functions and addressing life‐altering maladies such as cardiac disorders. Systems that incorporate both modulation of cellular function and recording capabilities can enhance the utility of these approaches and their customization to the needs of each patient. Here we report an integrated optogenetic and bioelectronic platform for stable and long‐term stimulation and monitoring of cardiomyocyte function in vitro. Optical inputs are achieved through the expression of a photoactivatable adenylyl cyclase, that when irradiated with blue light causes a dose‐dependent and time‐limited increase in the secondary messenger cyclic adenosine monophosphate with subsequent rise in autonomous cardiomyocyte beating rate. Bioelectronic readouts are obtained through a multi‐electrode array that measures real‐time electrophysiological responses at 32 spatially‐distinct locations. Irradiation at 27 µW mm−2 results in a 14% elevation of the beating rate within 20–25 min, which remains stable for at least 2 h. The beating rate can be cycled through "on" and "off" light states, and its magnitude is a monotonic function of irradiation intensity. The integrated platform can be extended to stretchable and flexible substrates, and can open new avenues in bioelectronic medicine, including closed‐loop systems for cardiac regulation and intervention, for example, in the context of arrythmias. [ABSTRACT FROM AUTHOR]

Melanosomal pH is important for the synthesis of melanin as the rate‐limiting enzyme, tyrosinase, is very pH‐sensitive. The soluble adenylyl cyclase (sAC) signaling pathway was recently identified as a regulator of melanosomal pH in melanocytes; however, the melanosomal proteins critical for sAC‐dependent regulation of melanosomal pH were undefined. We now systematically examine four well‐characterized melanosomal membrane proteins to determine whether any of them are required for sAC‐dependent regulation of melanosomal pH. We find that OA1, OCA2, and SLC45A2 are dispensable for sAC‐dependent regulation of melanosomal pH. In contrast, TPC2 activity is required for sAC‐dependent regulation of melanosomal pH and melanin synthesis. In addition, activation of TPC2 by NAADP‐AM rescues melanosomal pH alkalinization and reduces melanin synthesis following pharmacologic or genetic inhibition of sAC signaling. These studies establish TPC2 as a critical melanosomal protein for sAC‐dependent regulation of melanosomal pH and pigmentation. [ABSTRACT FROM AUTHOR]

Background: The PG1037 gene is part of the uvrA‐PG1037‐pcrA operon in Porphyromonas gingivalis. It encodes for a protein of unknown function upregulated under hydrogen peroxide (H2O2)‐induced oxidative stress. Bioinformatic analysis shows that PG1037 has a zinc‐finger motif, two peroxidase motifs, and one cytidylate kinase domain. The aim of this study is to characterize further the role of the PG1037 recombinant protein in the unique 8‐oxoG repair system in P. gingivalis. Materials and Methods: PG1037 recombinant proteins with deletions in the zinc‐finger or peroxidase motifs were created. Electrophoretic mobility shift assays were used to evaluate the ability of the recombinant proteins to bind 8‐oxoG‐containing oligonucleotides. Zinc binding, peroxidase, and Fenton reaction assays were used to assess the functional roles of the rPG1037 protein. A bacterial adenylate cyclase two‐bride assay was used to identify the partner protein of PG1037 in the repair of 8‐oxoG. Results: The recombinant PG1037 (rPG1037) protein carrying an N‐terminal His‐tag demonstrated an ability to recognize and bind 8‐oxoG‐containing oligonucleotide. In contrast to the wild‐type rPG1037 protein, the zinc‐finger motif deletion resulted in the loss of zinc and 8‐oxoG binding activities. A deletion of the peroxidase motif‐1 showed a decrease in peroxidase activity. Using a bacterial adenylate cyclase two‐hybrid system, there was no observed protein‐protein interaction of PG1037 with UvrA (PG1036), PcrA (PG1038), or mismatch repair system proteins. Conclusions: Taken together, the results show that PG1037 is an important member of a novel mechanism that recognizes and repairs oxidative stress‐induced DNA damage in P. gingivalis. [ABSTRACT FROM AUTHOR]

When mechanical stimulation was applied to free swimming Paramecium, forward swimming velocity transiently increased due to activation of the posterior mechanosensory channels. The behavior response, known as “escape response,” requires membrane hyperpolarization and the activation of K‐channel type adenylate cyclases. Our hypothesis is that this escape response also involves activation of hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels. HCN channels are activated by hyperpolarization and are modulated by cyclic nucleotides such as cAMP and cGMP. They play a critical role in many excitable cells in higher animals. If HCN channels act in Paramecium, this should help to enhance and prolong hyperpolarization, thereby increasing the swimming speed of Paramecium. This study used RNAi to examine the role of the HCN channel 1 in the escape responses by generating hcn1‐gene knockdown cells (hcn1‐KD). These cells showed reduced mechanically‐stimulated escape responses and a lack of cGMP‐dependent increases in swimming speed. Electrophysiological experiments demonstrated reduced hyperpolarization upon injection of large negative currents in hcn1‐KD cells. This is consistent with a decrease in HCN1 channel activity and changes in the escape response. These findings suggest that HCN1 channels are K+ channels that regulate the escape response of Paramecium by amplifying the hyperpolarizations elicited by posterior mechanical stimulation. [ABSTRACT FROM AUTHOR]

The melanocortin‐1 receptor (MC1R) is a G protein‐coupled receptor that plays a pivotal role in human skin pigmentation, melanin synthesis, redox homeostasis and inflammation. Loss‐of‐function MC1R variants suppress G protein‐coupled receptor coupling or cell surface expression leading to a decrease in adenyl cyclase activation and intracellular levels of cyclic adenosine monophosphate. Chronic activation of MC1R can occur in certain medical conditions such as Addison's disease and physiologic states such as pregnancy melasma. MC1R activation is more commonly caused by environmental exposure to ultraviolet (UV) radiation. Approved pharmacologic melanocortin agonists that activate MC1R signalling in a targeted manner or as a bystander effect have recently become available for erythropoietic protoporphyria, sexual desire disorders, monogenic obesity and syndromic obesity. Further, small peptide analogues of α–melanocortin‐stimulating hormone, human MC1R selective agonists, are photoprotective, decreasing the adverse impact of UV radiation (a primary risk factor for skin cancer) and are being investigated as potential chemoprevention strategies. MC1R activation through induction of UV‐protective skin pigmentation increased DNA repair, and control of aberrant cell growth may reduce the risk of melanoma but importantly does not prevent melanoma particularly in individuals with risk factors and regular skin examination remains critical in high‐risk individuals. [ABSTRACT FROM AUTHOR]

Heart failure with preserved ejection fraction (HFpEF) is one of the major subtypes of heart failure (HF) and no effective treatments for this common disease exist to date. Cardiac fibrosis is central to the pathology of HF and a potential avenue for the treatment of HFpEF. To explore key fibrosis‐related genes and pathways in the pathophysiological process of HFpEF, a mouse model of HFpEF was constructed. The relevant gene expression profiles were downloaded from the Gene Expression Omnibus database, and single‐sample Gene Set Enrichment Analysis (ssGSEA) was performed targeting fibrosis‐related pathways to explore differentially expressed genes (DEGs) in healthy control and HFpEF heart tissues with cross‐tabulation analysis of fibrosis‐related genes. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed on the identified fibrosis‐related genes. The two most significant DEGs were selected, and further validation was conducted in HFpEF mice. The results indicated that myocardial fibrosis was significantly upregulated in HFpEF mice compared to healthy controls, while the ssGSEA results revealed significant differences in the enrichment of nine fibrosis‐related pathways in HFpEF myocardial tissue, with 112 out of 798 DEGs being related to fibrosis. The in vivo results demonstrated that expression levels of resistin‐like molecule gamma (Relmg) and adenylate cyclase 1 (Adcy1) in the heart tissues of HFpEF mice were significantly higher and lower, respectively, compared to healthy controls. Taken together, these results suggest that Relmg and Acdy1 as well as the fibrosis process may be potential targets for HFpEF treatment. [ABSTRACT FROM AUTHOR]

Early life stress (ELS) exposure alters stress susceptibility in later life and affects vulnerability to stress‐related disorders, but how ELS changes the long‐lasting responsiveness of the stress system is not well understood. Zebrafish provides an opportunity to study conserved mechanisms underlying the development and function of the stress response that is regulated largely by the neuroendocrine hypothalamus–pituitary–adrenal/interrenal (HPA/I) axis, with glucocorticoids (GC) as the final effector. In this study, we established a method to chronically elevate endogenous GC levels during early life in larval zebrafish. To this end, we employed an optogenetic actuator, beggiatoa photoactivated adenylyl cyclase, specifically expressed in the interrenal cells of zebrafish and demonstrate that its chronic activation leads to hypercortisolaemia and dampens the acute‐stress evoked cortisol levels, across a variety of stressor modalities during early life. This blunting of stress–response was conserved in ontogeny at a later developmental stage. Furthermore, we observe a strong reduction of proopiomelanocortin (pomc)‐expression in the pituitary as well as upregulation of fkbp5 gene expression. Going forward, we propose that this model can be leveraged to tease apart the mechanisms underlying developmental programming of the HPA/I axis by early‐life GC exposure and its implications for vulnerability and resilience to stress in adulthood. [ABSTRACT FROM AUTHOR]

In this study, we investigated the roles of ROCK1 in regulating structural and functional features of caveolae located at the cell membrane of cardiomyocytes, adipocytes, and mouse embryonic fibroblasts (MEFs) as well as related physiopathological effects. Caveolae are small bulb‐shaped cell membrane invaginations, and their roles have been associated with disease conditions. One of the unique features of caveolae is that they are physically linked to the actin cytoskeleton that is well known to be regulated by RhoA/ROCKs pathway. In cardiomyocytes, we observed that ROCK1 deficiency is coincident with an increased caveolar density, clusters, and caveolar proteins including caveolin‐1 and ‐3. In the mouse cardiomyopathy model with transgenic overexpressing Gαq in myocardium, we demonstrated the reduced caveolar density at cell membrane and reduced caveolar protein contents. Interestingly, coexisting ROCK1 deficiency in cardiomyocytes can rescue these defects and preserve caveolar compartmentalization of β‐adrenergic signaling molecules including β1‐adrenergic receptor and type V/VI adenylyl cyclase. In cardiomyocytes and adipocytes, we detected that ROCK1 deficiency increased insulin signaling with increased insulin receptor activation in caveolae. In MEFs, we identified that ROCK1 deficiency increased caveolar and total levels of caveolin‐1 and cell membrane repair ability after mechanical or chemical disruptions. Together, these results demonstrate that ROCK1 can regulate caveolae plasticity and multiple functions including compartmentalization of signaling molecules and cell membrane repair following membrane disruption by mechanical force and oxidative damage. These findings provide possible molecular insights into the beneficial effects of ROCK1 deletion/inhibition in cardiomyocytes, adipocytes, and MEFs under certain diseased conditions. [ABSTRACT FROM AUTHOR]

Background and Purpose: Little is known of how cranial autonomic symptoms (CAS) in cluster headache and migraine may contribute to their severe headache phenotype. This strong association suggests the involvement of the cranial parasympathetic efferent pathway. To investigate its contribution, we studied the role of pituitary adenylate cyclase activating polypeptide‐38 (PACAP‐38), a potent sensory and parasympathetic neuropeptide, in modulating pre‐ and post‐ganglionic cranial parasympathetic projection neurons, and their influence on headache‐related trigeminal‐autonomic responses. Experimental Approach: Using PACAP‐38 and PACAP‐38 responsive receptor antagonists, electrophysiological, behavioural and facial neurovascular‐blood flow was measured in rats to probe trigeminal‐ and parasympathetic‐neuronal, periorbital thresholds and cranial‐autonomic outcomes, as they relate to primary headaches. Key Results: Sumatriptan attenuated the development of PACAP‐38 mediated activation and sensitization of trigeminocervical neurons and related periorbital allodynia. PACAP‐38 also caused activation and enhanced responses of dural‐responsive pre‐ganglionic pontine‐superior salivatory parasympathetic neurons. Further, the PACAP‐38 responsive receptor antagonists dissected a role of VPAC1 and PAC1 receptors in attenuating cranial‐autonomic and trigeminal‐neuronal responses to activation of the cranial parasympathetic projection, which requires post‐ganglionic parasympathetic neurotransmission. Conclusion and Implications: Given the prevailing view that sumatriptan acts to some degree via a peripheral mechanism, our data support that PACAP‐38 mediated receptor activation modulates headache‐related cranial‐autonomic and trigeminovascular responses via peripheral and central components of the cranial parasympathetic projection. This provides a mechanistic rationale for the association of CAS with more severe headache phenotypes in cluster headache and migraine, and supports the cranial parasympathetic projection as a potential novel locus for treatment by selectively targeting PACAP‐38 or PACAP‐38 responsive VPAC1/PAC1 receptors. [ABSTRACT FROM AUTHOR]

Cyanobacteria utilize CO2 and HCO3− as inorganic carbon (Ci) sources. In low Ci, like in ambient air, cyanobacteria efficiently collect Ci using a carbon concentrating mechanism (CCM). The CCM includes bicarbonate transporters SbtA, BicA and BCT1; the specialized NDH complexes NDH‐13 and NDH‐14, which convert CO2 to HCO3− in the cytoplasm; and carboxysomes that are protein shell encapsulated ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCo) and carbonic anhydrase containing bodies in which the first reaction of carbon fixation occurs. Ci‐dependent regulation of bicarbonate transporters and specialized NDH complexes, especially the regulation of the SbtA transporter, are well understood. CcmR (also called NdhR), CyAbrB2, CmpR and RbcR act as transcription factors regulating CCM genes. Ci signalling molecules detecting the metabolic status of the cells include 2‐oxoglutarate, which accumulates when the Ci/nitrogen ratio of the cell is high, and 2‐phosphoglycolate, the first intermediate of the photorespiration pathway, whose accumulation indicates low Ci. These signalling molecules act as corepressors and coactivators of the CcmR repressor protein, whereas 2‐phosphoglycolate and ribulose‐1,5‐bisphosphate activate transcription activator CmpR. In addition, bicarbonate or CO2 activates the adenylyl cyclase that produces cAMP, and ATP/ADP/AMP provide information about the energy status of the cell. Less is known about the molecular mechanisms regulating carboxysome dynamics or how production, activity and degradation of photosynthetic complexes are regulated by prevailing Ci conditions or which mechanisms adjust cell division according to Ci. This minireview summarizes the present knowledge about molecular mechanisms regulating cyanobacterial acclimation to prevailing Ci. [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]

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]

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]

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]

Background: Adenylyl cyclase 5 (ADCY5)‐related movement disorder (ADCY5‐RMD) is a rare, childhood‐onset disease resulting from pathogenic variants in the ADCY5 gene. The clinical features, diagnostic options, natural history, and treatments for this disease are poorly characterized and have never been established through a structured approach. Objective: This scoping review attempts to summarize all available clinical literature on ADCY5‐RMD. Methods: Eighty‐seven articles were selected for inclusion in this scoping review. The majority of articles identified were case reports or case series. Results: These articles demonstrate that patients with ADCY5‐RMD suffer from permanent and/ or paroxysmal hyperkinetic movements. The paroxysmal episodes can be worsened by environmental triggers, in particular the sleep–wake transition phase in the early morning. Occurrence of nocturnal paroxysmal dyskinesias and perioral twitches are highly suggestive of the diagnosis when present. In the majority of patients intellectual capacity is preserved. ADCY5‐RMD is considered a non‐progressive disorder, with inter‐individual variations in evolution with aging. Somatic mosaicism, mode of inheritance and the location of the mutation within the protein can influence phenotype. Conclusions: The current evidence for therapeutic options for ADCY5‐RMD is limited: caffeine, benzodiazepines and deep brain stimulation have been consistently reported to be useful in case reports and case series. [ABSTRACT FROM AUTHOR]

Cyclic AMP (cAMP) has a key role in psoriasis pathogenesis, as indicated by the therapeutic efficacy of phosphodiesterase inhibitors that prevent the degradation of cAMP. However, whether soluble adenylate cyclase (sAC) (encoded by the ADCY10 gene), which is an important source for cAMP, is involved in Th17 cell‐mediated inflammation or could be an alternative therapeutic target in psoriasis is unknown. We have utilized the imiquimod model of murine psoriasiform dermatitis to address this question. Adcy10−/− mice had reduced erythema, scaling and swelling in the skin and reduced CD4+ IL17+ cell numbers in the draining lymph nodes, compared with wild‐type mice after induction of psoriasiform dermatitis with imiquimod. Keratinocyte‐specific knock out of Adcy10 had no effect on imiquimod‐induced ear swelling suggesting keratinocyte sAC has no role in imiquimod‐induced inflammation. During Th17 polarization in vitro, naive T cells from Adcy10−/− mice exhibited reduced IL17 secretion and IL‐17+ T‐cell proliferation suggesting that differentiation into Th17 cells is suppressed without sAC activity. Interestingly, loss of sAC did not impact the expression of Th17 lineage‐defining transcription factors (such as Rorc and cMaf) but rather was required for CREB‐dependent gene expression, which is known to support Th17 cell gene expression. Finally, topical application of small molecule sAC inhibitors (sACi) reduced imiquimod‐induced psoriasiform dermatitis and Il17 gene expression in the skin. Collectively, these findings demonstrate that sAC is important for psoriasiform dermatitis in mouse skin. sACi may provide an alternative class of topical therapeutics for Th17‐mediated skin diseases. [ABSTRACT FROM AUTHOR]

Septin‐based ring complexes maintain the sperm annulus. Defective annular structures are observed in the sperm of Sept12‐ and Sept4‐null mice. In addition, sperm capacitation, a process required for proper fertilization, is inhibited in Sept4‐null mice, implying that the sperm annulus might play a role in controlling sperm capacitation. Hence, we analyzed sperm capacitation of sperm obtained from SEPT12 Ser196 phosphomimetic (S196E), phosphorylation‐deficient (S196A), and SEPT4‐depleted mutant mice. Capacitation was reduced in the sperm of both the Sept12 S196E‐ and Sept12 S196A‐knock‐in mice. The protein levels of septins, namely, SEPT4 and SEPT12, were upregulated, and these proteins were concentrated in the sperm annulus during capacitation. Importantly, the expression of soluble adenylyl cyclase (sAC), a key enzyme that initiates capacitation, was upregulated, and sAC was recruited to the sperm annulus following capacitation stimulation. We further found that SEPT12, SEPT4, and sAC formed a complex and colocalized to the sperm annulus. Additionally, sAC expression was reduced and disappeared in the annulus of the SEPT12 S196E‐ and S196A‐mutant mouse sperm. In the sperm of the SEPT4‐knockout mice, sAC did not localize to the annulus. Thus, our data demonstrate that SEPT12 phosphorylation status and SEPT4 activity jointly regulate sAC protein levels and annular localization to induce sperm capacitation. [ABSTRACT FROM AUTHOR]

The cyclic adenosine monophosphate‐protein kinase A (cAMP‐PKA) signalling pathway is evolutionarily conserved in eukaryotes and plays a crucial role in defending against external environmental challenges, which can modulate the cellular response to external stimuli. Arthrobotrys oligospora is a typical nematode‐trapping fungus that specializes in adhesive networks to kill nematodes. To elucidate the biological roles of the cAMP‐PKA signalling pathway, we characterized the orthologous adenylate cyclase AoAcy, a regulatory subunit (AoPkaR), and two catalytic subunits (AoPkaC1 and AoPkaC2) of PKA in A. oligospora by gene disruption, transcriptome, and metabolome analyses. Deletion of Aoacy significantly reduced the levels of cAMP and arthrobotrisins. Results revealed that Aoacy, AopkaR, and AopkaC1 were involved in hyphal growth, trap morphogenesis, sporulation, stress resistance, and autophagy. In addition, Aoacy and AopkaC1 were involved in the regulation of mitochondrial morphology, thereby affecting energy metabolism, whereas AopkaC2 affected sporulation, nuclei, and autophagy. Multi‐omics results showed that the cAMP‐PKA signalling pathway regulated multiple metabolic and cellular processes. Collectively, these data highlight the indispensable role of cAMP‐PKA signalling pathway in the growth, development, and pathogenicity of A. oligospora, and provide insights into the regulatory mechanisms of signalling pathways in sporulation, trap formation, and lifestyle transition. [ABSTRACT FROM AUTHOR]

Adenosine‐3′, 5′‐cyclic monophosphate (cAMP) produced by adenylate cyclases (ADCYs) is an established key regulator of cell homoeostasis. However, its role in cell cycle control is still controversially discussed. This study focussed on the impact of soluble HCO3− ‐activated ADCY10 on cell cycle progression. Effects are quantified with Bayesian inference integrating a mathematical model and experimental data. The activity of ADCY10 in human umbilical vein endothelial cells (HUVECs) was either pharmacologically inhibited by KH7 or endogenously activated by HCO3−. Cell numbers of individual cell cycle phases were assessed over time using flow cytometry. Based on these numbers, cell cycle dynamics were analysed using a mathematical model. This allowed precise quantification of cell cycle dynamics with model parameters that describe the durations of individual cell cycle phases. Endogenous inactivation of ADCY10 resulted in prolongation of mean cell cycle times (38.7 ± 8.3 h at 0 mM HCO3− vs 30.3 ± 2.7 h at 24 mM HCO3−), while pharmacological inhibition resulted in functional arrest of cell cycle by increasing mean cell cycle time after G0/G1 synchronization to 221.0 ± 96.3 h. All cell cycle phases progressed slower due to ADCY10 inactivation. In particular, the G1‐S transition was quantitatively the most influenced by ADCY10. In conclusion, the data of the present study show that ADCY10 is a key regulator in cell cycle progression linked specifically to the G1‐S transition. [ABSTRACT FROM AUTHOR]

As a kind of xanthine alkaloid, caffeine is widely present in beverages, food, and analgesic drugs. Our previous studies have shown that prenatal caffeine exposure (PCE) can induce programmed hypersensitivity of the hypothalamic–pituitary–adrenal (HPA) axis in offspring rats, which is involved in developing many chronic adult diseases. The present study further examined the potential molecular mechanism and toxicity targets of hippocampal dysfunction, which might mediate the programmed hypersensitivity of the HPA axis in offspring. Pregnant rats were intragastrically administered with 0, 30, and 120 mg/kg/day caffeine from gestational days (GD) 9–20, and the fetal rats were extracted at GD20. Rat fetal hippocampal H19‐7/IGF1R cell line was treated with caffeine, adenosine A2A receptor (A2AR) agonist (CGS‐21680) or adenylate cyclase agonist (forskolin) plus caffeine. Compared with the control group, hippocampal neurons of male fetal rats by PCE displayed increased apoptosis and reduced synaptic plasticity, whereas glutamate decarboxylase 67 (GAD67) expression was increased. Moreover, the expression of A2AR was down‐regulated, PCE inhibited the cAMP/PKA/CREB/BDNF/TrkB pathway. Furthermore, the results in vitro were consistent with the in vivo study. Both CGS21680 and forskolin could reverse the above alteration caused by caffeine. These results indicated that PCE inhibits the BDNF pathway and mediates the hippocampus's glutamate (Glu) excitotoxicity. The compensatory up‐regulation of GAD67 unbalanced the Glu/gamma‐aminobutyric acid (GABA)ergic output, leading to the impaired negative feedback to the hypothalamus and hypersensitivity of the HPA axis. [ABSTRACT FROM AUTHOR]

Recent biochemical experiments have indicated that in Candida albicans, a commensal fungal pathogen, the Ras signaling pathway plays a significant role in the yeast‐to‐hyphal transition; specifically, two enzymes in this pathway, Adenyl Cyclase Cyr1 and GTPase activating protein Ira2, facilitate this transition, in the presence of energy sensor ATP. However, the precise mechanism by which protein interactions between Ira2 and Cyr1 and the energy sensor ATP result in the yeast‐to‐hyphal transition and create a switch‐like process are unknown. We propose a new set of biochemical reaction steps that captures all the essential interactions between Ira2, Cyr1, and ATP in the Ras pathway. With the help of chemical reaction network theory, we demonstrate that this set of biochemical reaction steps results in bistability. Further, bifurcation analysis of the differential equations based on this set of reaction steps supports the existence of a bistable switch, and this switch may act as a checkpoint mechanism for the promotion of growth‐to‐hyphal transition in C. albicans. [ABSTRACT FROM AUTHOR]

Tau is a protein that normally participates in the assembly and stability of microtubules. However, it can form intraneuronal hyperphosphorylated aggregates that are hallmarks of Alzheimer's disease and other neurodegenerative disorders known as tauopathies. Tau can be phosphorylated by multiple kinases at several sites. Among such kinases, the cAMP‐dependent protein kinase A (PKA) phosphorylates tau at Ser214 (pTAU‐S214), an event that was shown to reduce the pathological assembly of the protein. Given that the neuronal cAMP/PKA‐activated cascade is involved in synaptic plasticity and memory, and that cAMP‐enhancing strategies demonstrated promising therapeutic potential for the treatment of cognitive deficits, we investigated the impact of cAMP on pTAU‐S214 in N2a cells and rat hippocampal slices. Our results confirm that the activation of adenylyl cyclase increases pTAU‐S214 in both model systems and, more interestingly, this effect is mimicked by GEBR‐7b, a phosphodiesterase 4D inhibitor with proven pro‐cognitive efficacy in rodents. [ABSTRACT FROM AUTHOR]

The sand fly Lutzomyia longipalpis is the main vector of Leishmania infantum in the Americas. Female sand flies ingest sugar‐rich solutions and blood, which are digested in the midgut. Digestion of nutrients is an essential function performed by digestive enzymes, which require appropriate physiological conditions. One of the main aspects that influence enzymatic activity is the gut pH, which must be tightly controlled. Considering second messengers are frequently involved in the coordination of tightly regulated physiological events, we investigated if the second messenger cAMP would participate in the process of alkalinization in the abdominal midgut of female L. longipalpis. In midguts containing the indicator dye bromothymol‐blue, cAMP stimulated the alkalinization of the midgut lumen. Through another technique based on the use of fluorescein as a pH indicator, we propose that cAMP is involved in the alkalinization of the midgut by activating HCO3– transport from the enterocyte's cytoplasm to the lumen. The results strongly suggested that the carrier responsible for this process would be a HCO3−/Cl− antiporter located in the enterocytes' apical membrane. Hematophagy promotes the release of alkalinizing hormones in the hemolymph; however, when the enzyme adenylyl cyclase, responsible for cAMP production, was inhibited, we observed that the hemolymph from blood‐fed L. longipalpis' females did not stimulate midgut alkalinization. This result indicated that hormone‐stimulated alkalinization is mediated by cAMP. In the present study, we provide evidences that cAMP has a key role in the control of intestinal pH. [ABSTRACT FROM AUTHOR]

Exercise increases the muscular secretion of Interleukin‐6 (IL‐6), which is partially regulated by β2‐adrenergic receptor signaling. Nobiletin is a polymethoxyflavone (PMF) found in citrus fruits that induces the secretion of IL‐6 from C2C12 myotubes, but it remains unclear whether nobiletin promotes IL‐6 secretion during exercise. The aim of this study was to clarify the effects of nobiletin on IL‐6 secretion during exercise. Nobiletin and epinephrine were found to synergistically increase IL‐6 secretion from differentiated C2C12 cells, which was suppressed by the inhibition of adenylyl cyclase (AC) or protein kinase A (PKA). Treadmill running for 60 min increased plasma levels of IL‐6, epinephrine, and norepinephrine in rats. Nobiletin (5 mg/kg) orally administered 30 min before running increased plasma IL‐6 levels further, although it did not increase plasma epinephrine and norepinephrine. In a similar manner to IL‐6, nobiletin and epinephrine synergistically increased the secretion of C‐X‐C motif chemokine ligand 1 (CXCL‐1) from C2C12 cells, or the increase in plasma CXCL‐1 was enhanced by nobiletin after treadmill running of rats. Our results suggest that nobiletin promotes IL‐6 and CXCL‐1 secretion from skeletal muscle by synergistic enhancement of the PKA pathway in β2‐adrenergic receptor signaling. [ABSTRACT FROM AUTHOR]

For at least 50 years, the prevailing view has been that the adenylate cyclase (AC)/cyclic AMP (cAMP)/protein kinase A pathway is the predominant signal mediating the hepatic glucose‐mobilizing actions of glucagon. A wealth of evidence, however, supports the alternative, that the operative signal most of the time is the phospholipase C (PLC)/inositol‐phosphate (IP3)/calcium/calmodulin pathway. The evidence can be summarized as follows: (1) The consensus threshold glucagon concentration for activating AC ex vivo is 100 pM, but the statistical hepatic portal plasma glucagon concentration range, measured by RIA, is between 28 and 60 pM; (2) Within that physiological concentration range, glucagon stimulates the PLC/IP3 pathway and robustly increases glucose output without affecting the AC/cAMP pathway; (3) Activation of a latent, amplified AC/cAMP pathway at concentrations below 60 pM is very unlikely; and (4) Activation of the PLC/IP3 pathway at physiological concentrations produces intracellular effects that are similar to those produced by activation of the AC/cAMP pathway at concentrations above 100 pM, including elevated intracellular calcium and altered activities and expressions of key enzymes involved in glycogenolysis, gluconeogenesis, and glycogen synthesis. Under metabolically stressful conditions, as in the early neonate or exercising adult, plasma glucagon concentrations often exceed 100 pM, recruiting the AC/cAMP pathway and enhancing the activation of PLC/IP3 pathway to boost glucose output, adaptively meeting the elevated systemic glucose demand. Whether the AC/cAMP pathway is consistently activated in starvation or diabetes is not clear. Because the importance of glucagon in the pathogenesis of diabetes is becoming increasingly evident, it is even more urgent now to resolve lingering uncertainties and definitively establish glucagon's true mechanism of glycemia regulation in health and disease. [ABSTRACT FROM AUTHOR]

Summary: Light is the crucial environmental signal for desiccation‐tolerant cyanobacteria to activate photosynthesis and prepare for desiccation at dawn. However, the photobiological characteristics of desert cyanobacteria adaptation to one of the harshest habitats on Earth remain unresolved. In this study, we surveyed the genome of a subaerial desert cyanobacterium Nostoc flagelliforme and identified two phytochromes and seven cyanobacteriochromes (CBCRs) with one or more bilin‐binding GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domains. Biochemical and spectroscopic analyses of 69 purified GAF‐containing proteins from recombinant phycocyanobilin (PCB), biliverdin or phycoerythrobilin‐producing Escherichia coli indicated that nine of these proteins bind chromophores. Further investigation revealed that 11 GAFs form covalent adducts responsive to near‐UV and visible light: eight GAFs contained PCB chromophores, three GAFs contained biliverdin chromophores and one contained the PCB isomer, phycoviolobilin. Interestingly, COO91_03972 is the first‐ever reported GAF‐only CBCR capable of sensing five wavelengths of light. Bioinformatics and biochemical analyses revealed that residue P132 of COO91_03972 is essential for chromophore binding to dual‐cysteine CBCRs. Furthermore, the complement of N. flagelliforme CBCRs is enriched in red light sensors. We hypothesize that these sensors are critical for the acclimatization of N. flagelliforme to weak light environments at dawn. [ABSTRACT FROM AUTHOR]

Activation of adenylyl cyclases (ACs) by G‐protein Gαs catalyzes the production of cyclic adenosine monophosphate (cAMP), a key second messenger that regulates diverse physiological responses. There are 10 AC isoforms present in humans, with AC5 and AC6 proposed to play vital roles in cardiac function. We have previously shown that under hypoxic conditions, AC6 is amenable to post‐translational modification by nitrosylation, resulting in decreased AC catalytic activity. Using a computational model of the AC6–Gαs complex, we predicted key nitrosylation‐amenable cysteine residues involved in the interaction of AC6 with Gαs and pursued a structure–function analysis of these cysteine residues in both AC6 and Gαs. Our results based on site‐directed mutagenesis of AC6 and Gαs, a constitutively active Gαs, AC activity, and live cell intracellular cAMP assays suggest that Cys1004 in AC6 (subunit C2) and Cys237 in Gαs are present at the AC–Gαs interface and are important for the activation of AC6 by Gαs. We further provide mechanistic evidence to show that mutating Cys 1004 in the second catalytic domain of AC6 makes it amenable to inhibition by Gαi, which may account for decreased functional activity of AC6 when this residue is unavailable. [ABSTRACT FROM AUTHOR]

β2‐Adrenoceptor (β2‐AR) signaling decreases the transcriptional activity of forkhead box O (FoxO), but the underlying mechanisms remain incompletely understood. Here, we investigated how β2‐AR signaling regulates the protein abundance of FoxO and its transcriptional activity in skeletal muscle. We observed that stimulation of β2‐AR with its selective agonist, clenbuterol, rapidly decreased FoxO1 mRNA expression, and this was accompanied by a decrease in either FoxO1 protein level or FoxO transcriptional activity. We subsequently observed that miR‐374b‐5p and miR‐7a‐1‐3p were rapidly upregulated in response to β2‐AR stimulation. Transfection with mimics of either miRNA successfully decreased FoxO1 mRNA. Moreover, because β2‐AR stimulation increased cAMP concentration, pretreatment with an adenylyl cyclase inhibitor canceled out these effects of β2‐AR stimulation. These results suggest that β2‐AR stimulation results in rapid upregulation of miR‐374b‐5p and miR‐7a‐1‐3p in myotubes, which in turn results in a decrease in FoxO1 mRNA expression via the β2‐AR–cAMP signaling pathway. [ABSTRACT FROM AUTHOR]

Aims: The α2‐adrenergic receptor (α2‐AR) agonists have been shown to be effective in the treatment of various pain. For example, dexmedetomidine (DEX), a selective α2A‐AR agonist, can be used for peripheral analgesia. However, it is not yet fully elucidated for the precise molecular mechanisms. P2X3 receptor is a major receptor processing nociceptive information in primary sensory neurons. Herein, we show that a functional interaction of α2A‐ARs and P2X3 receptors in dorsal root ganglia (DRG) neurons could contribute to peripheral analgesia of DEX. Methods: Electrophysiological recordings were carried out on rat DRG neurons, and nociceptive behavior was quantified in rats. Results: The activation of α2A‐ARs by DEX suppressed P2X3 receptor‐mediated and α,β‐methylene‐ATP (α,β‐meATP)‐evoked inward currents in a concentration‐dependent and voltage‐independent manner. Pre‐application of DEX shifted the α,β‐meATP concentration‐response curve downwards, with a decrease of 50.43 ± 4.75% in the maximal current response of P2X3 receptors to α,β‐meATP in the presence of DEX. Suppression of α,β‐meATP‐evoked currents by DEX was blocked by the α2A‐AR antagonist BRL44408 and prevented by intracellular application of the Gi/o protein inhibitor pertussis toxin, the adenylate cyclase activator forskolin, and the cAMP analog 8‐Br‐cAMP. DEX also suppressed α,β‐meATP‐evoked action potentials through α2A‐ARs in rat DRG neurons. Finally, the activation of peripheral α2A‐ARs by DEX had an analgesic effect on the α,β‐meATP‐induced nociception. Conclusions: These results suggested that activation of α2A‐ARs by DEX suppressed P2X3 receptor‐mediated electrophysiological and behavioral activity via a Gi/o proteins and cAMP signaling pathway, which was a novel potential mechanism underlying analgesia of peripheral α2A‐AR agonists. [ABSTRACT FROM AUTHOR]

Neuronal primary cilia are crucial for body weight maintenance. Type III adenylyl cyclase (AC3) is abundantly enriched in neuronal cilia, and mice with global AC3 ablation are obese. However, whether AC3 regulates body weight through its ciliary expression and the mechanism underlying this potential regulation are not clear. In this study, humanized AC3 knock‐in mice that are resistant to high‐fat diet (HFD)‐induced obesity are generated, and increases in the number and length of cilia in the ventromedial hypothalamus (VMH) are shown. It is demonstrated that mice with specifically knocked down ciliary AC3 expression in the VMH show pronounced HFD‐induced obesity. In addition, in vitro and in vivo analyses of the VMH show that ciliary AC3 regulates autophagy by binding an autophagy‐related gene, gamma‐aminobutyric acid A receptor‐associated protein (GABARAP). Mice with GABARAP knockdown in the VMH exhibit exacerbated HFD‐induced obesity. Overall, the findings may reveal a potential mechanism by which ciliary AC3 expression regulates body weight in the mouse VMH. [ABSTRACT FROM AUTHOR]

Hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels function as pacemaker channels in spontaneously active cells. We studied the existence of HCN channels and their functional roles in the interstitial cells of Cajal (ICC) from the mouse colon using electrophysiological, immunohistochemical and molecular techniques. HCN1 and HCN3 channels were detected in anoctamin‐1 (Ca2+‐activated Cl− channel; ANO1)‐positive cells within the muscular and myenteric layers in colonic tissues. The mRNA transcripts of HCN1 and HCN3 channels were expressed in ANO1‐positive ICC. In the deletion of HCN1 and HCN3 channels in colonic ICC, the pacemaking potential frequency was reduced. Basal cellular adenylate cyclase activity was decreased by adenylate cyclase inhibitor in colonic ICC, whereas cAMP‐specific phosphodiesterase inhibitors increased it. 8‐Bromo‐cyclic AMP and rolipram increased spontaneous intracellular Ca2+ oscillations. In addition, Ca2+‐dependent adenylate cyclase 1 (AC1) mRNA was detected in colonic ICC. Sulprostone, a PGE2‐EP3 agonist, increased the pacemaking potential frequency, maximum rate of rise of resting membrane in pacemaker potentials and basal cellular adenylate cyclase activity in colonic ICC. These results indicate that HCN channels exist in colonic ICC and participate in generating pacemaking potentials. Thus, HCN channels may be therapeutic targets in disturbed colonic motility disorders. [ABSTRACT FROM AUTHOR]

Several toxins acting on animal cells present different, but specific, interactions with cholesterol. Bordetella pertussis infects the human respiratory tract and causes whooping cough, a highly contagious and resurgent disease. Its virulence factor adenylate cyclase toxin (ACT) plays an important role in the course of infection. ACT is a pore‐forming cytolysin belonging to the Repeats in ToXin (RTX) family of leukotoxins/hemolysins and is capable of permeabilizing several cell types and lipid vesicles. Previously, we observed that in the presence of cholesterol ACT induces greater liposome permeabilization. Similarly, recent reports also implicate cholesterol in the cytotoxicity of an increasing number of pore‐forming RTX toxins. However, the mechanistic details by which this sterol promotes the lytic activity of ACT or of these other RTX toxins remain largely unexplored and poorly understood. Here, we have applied a combination of biophysical techniques to dissect the role of cholesterol in pore formation by ACT. Our results indicate that cholesterol enhances the lytic potency of ACT by promoting toxin oligomerization, a step which is indispensable for ACT to accomplish membrane permeabilization and cell lysis. Since our experimental design eliminates the possibility that this cholesterol effect derives from toxin accumulation due to lateral lipid phase segregation, we hypothesize that cholesterol facilitates lytic pore formation, by favoring a toxin conformation more prone to protein–protein interactions and oligomerization. Our data shed light on the complex relationship between lipid membranes and protein toxins acting on these membranes. Coupling cholesterol binding, increased oligomerization and increased lytic activity is likely pertinent for other RTX cytolysins. [ABSTRACT FROM AUTHOR]

Thiazolidinediones are synthetic PPARγ ligands that enhance insulin sensitivity, and that could increase insulin secretion from β‐cells. However, the functional role and mechanism(s) of action in pancreatic β‐cells have not been investigated in detail. [ABSTRACT FROM AUTHOR]

Neuronatin (NNAT) is a transmembrane protein in the endoplasmic reticulum involved in metabolic regulation. It shares sequence homology with sarcolipin (SLN), which negatively regulates the sarco(endo)plasmic reticulum Ca2+‐ATPase (SERCA) that maintains energy homeostasis in muscles. Here, we examined whether NNAT could uncouple the Ca2+ transport activity of SERCA from ATP hydrolysis, similarly to SLN. NNAT significantly reduced Ca2+ uptake without altering SERCA activity, ultimately lowering the apparent coupling ratio of SERCA. This effect of NNAT was reversed by the adenylyl cyclase activator forskolin. Furthermore, soleus muscles from high fat diet (HFD)‐fed mice showed a significant downregulation in NNAT content compared with chow‐fed mice, whereas an upregulation in NNAT content was observed in fast‐twitch muscles from HFD‐ versus chow‐ fed mice. Therefore, NNAT is a SERCA uncoupler in cells and may function in adaptive thermogenesis. [ABSTRACT FROM AUTHOR]

High temperature constitutes a serious threat to crops. 3′,5′‐cyclic adenosine monophosphate (cAMP) is increasingly proved to play important roles in plants. However, the mechanism of cAMP‐mediated thermotolerance in root remains unclear. This study was designed to investigate cAMP roles in thermotolerance of maize root. Adenylyl cyclase (AC) catalyzes ATP to generate cAMP. For two identified maize AC genes, ZmRPP13‐LK3 and ZmPSiP, the predictive ABA response element was found in their promoter fragments. Both ABA and heat stress treatment increased the promoter activity of ZmRPP13‐LK3 and ZmPSiP. In the roots of maize ABA‐deficient mutant vp5, the heat‐caused increase of these two gene expression and cAMP content was obviously less than that in the roots of its wild‐type Vp5. cAMP pretreatment decreased H2O2 and malondialdehyde content, but increased the activities of superoxide dismutase and ascorbate peroxidase in roots compared to no pretreatment under heat stress. By iTRAQ proteomics analysis, 268 cAMP‐mediated differentially expressed proteins (DEPs) were identified from maize roots exposed to heat stress. The integrated analysis of these DEPs showed that cAMP was involved in many important biological processes, including ion uptake, protein degradation via autophagy or ubiquitin–proteasome system, a rapid delivery of stress‐related cargo molecules by vesicle trafficking, and adaptation response by defense proteins. To determine whether the gene expression of these DEPs was regulated by cAMP and ABA, the gene expression of randomly selected 15 DEPs as ion transporters and heat shock proteins was analyzed, and the results indicated that cAMP and ABA pretreatment also enhanced the expression of their genes. Additionally, the cAMP pretreatment increased Ca2+, PO43−, and NO3− uptake, but reduced K+ uptake in roots under heat stress. All above‐mentioned results suggest that ABA‐mediated cAMP signaling may contribute to better thermotolerance in maize roots. [ABSTRACT FROM AUTHOR]

Background: The type 3 adenylyl cyclase (AC3) enzyme is involved in the synthesis of cyclic adenosine monophosphate (cAMP). It is primarily expressed in the central nervous system (CNS) and plays a crucial role in neurogenesis and neural dendritic arborization. However, the AC3's functional role in the gastrointestinal tract remains ambiguous. Methods: AC3 expression in enteric tissue of AC3+/+ mice was investigated using immunohistochemistry and RT‐PCR. AC3 knock‐out mice (AC3−/−) were used to examine the effect of AC3 on the enteric nervous system (ENS) function and the number of cilia and apoptotic cells. Additionally, total gastrointestinal transit time and colonic motility were compared between the AC3−/− and AC3+/+ groups of mice. Key Results: AC3 was predominately expressed in the myenteric plexus of the large intestine. Colonic‐bead expulsion analysis showed accelerated propulsion in the large intestine of the AC3−/− mice. The AC3−/− mice demonstrated reduced nerve fibers and enteric glial cells count in colonic mucosa compared to the AC3+/+ mice. Furthermore, AC3−/− mice exhibited increased cellular apoptosis and reduced ARL13B+ cilium cells in the colonic lamina propria compared to the AC3+/+ mice. Conclusions: In AC3−/− mice, innervation of the lamina propria in the colonic mucosa was reduced and colonic propulsion was accelerated. AC3 is crucial for the development and function of the adult neural network of ENS. AC3 deficiency caused atrophy in the colonic mucosal neural network of mice. [ABSTRACT FROM AUTHOR]

Aims: The transient receptor potential melastin‐8 (TRPM8) channel is a "cooling" receptor expressed in primary sensory neurons and can be activated by compounds like menthol or icilin. TRPM8 is involved in the regulation of urinary bladder sensory function and contraction, but the role of TRPM8 in the ureter, particularly in the human ureter, is poorly understood. The aim of this study is to examine the effects of TRPM8 activation on human ureter contraction. Methods: Human ureters were acquired from 20 patients undergoing radical nephrectomy. Contractions of ureter strips were recorded by an isometric transducer in the organ bath. Ureteral TRPM8 expression in the human ureter was examined by immunofluorescence and western blot. Results: The two TRPM8 agonists menthol and icilin both reduced the frequency of spontaneous, electrical field stimulation, or neurokinin A‐evoked ureteral contractions in a dose‐dependent manner. The inhibitory effects were decreased by 10‐fold in mucosa‐denuded strips. The inhibitory effects of TRPM8 agonists were mimicked by calcitonin gene‐related peptide (CGRP), and were blocked by KRP2579 (a TRPM8 antagonist), tetrodotoxin (a sodium channel blocker), olcegepant (BIBN, a CGRP receptor antagonist), SQ22536 (an adenylate cyclase antagonist), or H89 (a nonspecific cAMP‐dependent protein kinase A inhibitor). TRPM8 was coexpressed with CGRP on the nerves located in the suburothelial and intermuscular regions and was not expressed in the urothelium. Conclusions: The TRPM8 channel expressed on sensory nerve terminals of the human ureter is involved in the inhibitory sensory neurotransmission and modulate ureter contraction via the CGRP–adenylyl cyclase–protein kinase A pathway. TRPM8 may be involved in stone‐induced changes in ureter contraction or pain. [ABSTRACT FROM AUTHOR]

Key points: In adult ventricular myocytes, the slow delayed rectifier (IKs) channels are distributed on the surface sarcolemma, not t‐tubules.In adult ventricular myocytes, KCNQ1 and KCNE1 have distinct cell surface and cytoplasmic pools.KCNQ1 and KCNE1 traffic from the endoplasmic reticulum to the plasma membrane by separate routes, and assemble into IKs channels on the cell surface.Liquid chromatography/tandem mass spectrometry applied to affinity‐purified KCNQ1 and KCNE1 interacting proteins reveals novel interactors involved in protein trafficking and assembly.Microtubule plus‐end binding protein 1 (EB1) binds KCNQ1 preferentially in its dimer form, and promotes KCNQ1 to reach the cell surface.An LQT1‐associated mutation, Y111C, reduces KCNQ1 binding to EB1 dimer. Slow delayed rectifier (IKs) channels consist of KCNQ1 and KCNE1. IKs functions as a 'repolarization reserve' in the heart by providing extra current for ventricular action potential shortening during β‐adrenergic stimulation. There has been much debate about how KCNQ1 and KCNE1 traffic in cells, where they associate to form IKs channels, and the distribution pattern of IKs channels relative to β‐adrenergic signalling complex. We used experimental strategies not previously applied to KCNQ1, KCNE1 or IKs, to provide new insights into these issues. 'Retention‐using‐selected‐hook' experiments showed that newly translated KCNE1 constitutively trafficked through the conventional secretory path to the cell surface. KCNQ1 largely stayed in the endoplasmic reticulum, although dynamic KCNQ1 vesicles were observed in the submembrane region. Disulphide‐bonded KCNQ1/KCNE1 constructs reported preferential association after they had reached cell surface. An in situ proximity ligation assay detected IKs channels in surface sarcolemma but not t‐tubules of ventricular myocytes, similar to the reported location of adenylate cyclase 9/yotiao. Fluorescent protein‐tagged KCNQ1 and KCNE1, in conjunction with antibodies targeting their extracellular epitopes, detected distinct cell surface and cytoplasmic pools of both proteins in myocytes. We conclude that, in cardiomyocytes, KCNQ1 and KCNE1 traffic by different routes to surface sarcolemma where they assemble into IKs channels. This mode of delayed channel assembly helps IKs fulfil its function of repolarization reserve. Proteomic experiments revealed a novel KCNQ1 interactor, microtubule plus‐end binding protein 1 (EB1). EB1 dimer (active form) bound KCNQ1 and increased its surface level. An LQT1 mutation, Y111C, reduced KCNQ1 binding to EB1 dimer. Key points: In adult ventricular myocytes, the slow delayed rectifier (IKs) channels are distributed on the surface sarcolemma, not t‐tubules.In adult ventricular myocytes, KCNQ1 and KCNE1 have distinct cell surface and cytoplasmic pools.KCNQ1 and KCNE1 traffic from the endoplasmic reticulum to the plasma membrane by separate routes, and assemble into IKs channels on the cell surface.Liquid chromatography/tandem mass spectrometry applied to affinity‐purified KCNQ1 and KCNE1 interacting proteins reveals novel interactors involved in protein trafficking and assembly.Microtubule plus‐end binding protein 1 (EB1) binds KCNQ1 preferentially in its dimer form, and promotes KCNQ1 to reach the cell surface.An LQT1‐associated mutation, Y111C, reduces KCNQ1 binding to EB1 dimer. [ABSTRACT FROM AUTHOR]

The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells are still a matter of intense research. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. The CyaA translocation region contains a segment, P454 (residues 454–484), which exhibits membrane‐active properties related to antimicrobial peptides. Herein, the results show that this peptide is able to translocate across membranes and to interact with calmodulin (CaM). Structural and biophysical analyses reveal the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrates that these residues play a crucial role in CyaA translocation into target cells. In addition, calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. It is proposed that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of CyaA by the CaM:P454 interaction in the cytosol may assist the entry of the N‐terminal catalytic domain by converting the stochastic motion of the polypeptide chain through the membrane into an efficient vectorial chain translocation into host cells. [ABSTRACT FROM AUTHOR]

Asthenozoospermia, defined by the absence or reduction of sperm motility, constitutes the most frequent cause of human male infertility. This pathological condition is caused by morphological and/or functional defects of the sperm flagellum, which preclude proper sperm progression. While in the last decade many causal genes were identified for asthenozoospermia associated with severe sperm flagellar defects, the causes of purely functional asthenozoospermia are still poorly defined. We describe here the case of an infertile man, displaying asthenozoospermia without major morphological flagellar anomalies and carrying a homozygous splicing mutation in SLC9C1 (sNHE), which we identified by whole‐exome sequencing. SLC9C1 encodes a sperm‐specific sodium/proton exchanger, which in mouse regulates pH homeostasis and interacts with the soluble adenylyl cyclase (sAC), a key regulator of the signalling pathways involved in sperm motility and capacitation. We demonstrate by means of RT‐PCR, immunodetection and immunofluorescence assays on patient's semen samples that the homozygous splicing mutation (c.2748 + 2 T > C) leads to in‐frame exon skipping resulting in a deletion in the cyclic nucleotide‐binding domain of the protein. Our work shows that in human, similar to mouse, SLC9C1 is required for sperm motility. Overall, we establish a homozygous truncating mutation in SLC9C1 as a novel cause of human asthenozoospermia and infertility. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is an X‐linked disease affecting male and rarely adult heterozygous females, resulting in death by the late 20s to early 30s. Previous studies reported depressed left ventricular function in DMD patients which may result from deranged intracellular Ca2+‐handling. To decipher the mechanism(s) underlying the depressed LV function, we tested the hypothesis that iPSC‐CMs generated from DMD patients feature blunted positive inotropic response to β‐adrenergic stimulation. To test the hypothesis, [Ca2+]i transients and contractions were recorded from healthy and DMD‐CMs. While in healthy CMs (HC) isoproterenol caused a prominent positive inotropic effect, DMD‐CMs displayed a blunted inotropic response. Next, we tested the functionality of the sarcoplasmic reticulum (SR) by measuring caffeine‐induced Ca2+ release. In contrast to HC, DMD‐CMs exhibited reduced caffeine‐induced Ca2+ signal amplitude and recovery time. In support of the depleted SR Ca2+ stores hypothesis, in DMD‐CMs the negative inotropic effects of ryanodine and cyclopiazonic acid were smaller than in HC. RNA‐seq analyses demonstrated that in DMD CMs the RNA‐expression levels of specific subunits of the L‐type calcium channel, the β1‐adrenergic receptor (ADRβ1) and adenylate cyclase were down‐regulated by 3.5‐, 2.8‐ and 3‐fold, respectively, which collectively contribute to the depressed β‐adrenergic responsiveness. [ABSTRACT FROM AUTHOR]

Thyroid hormones affect testicular development as well as functions like spermatogenesis and steroidogenesis, thereby influencing male fertility. Our group earlier showed that the stimulatory role of the thyroid hormone, T3, on the production of vascular endothelial growth factor (VEGF) by murine Leydig cells is mediated by steroids and hypoxia‐inducible factor‐1 (HIF‐1α). The current study further defines the signalling pathway(s) utilised by T3 to stimulate the production of steroids, VEGF and HIF‐1α in mouse Leydig tumour cell line (MLTC‐1). Specific inhibitors for different signalling molecules were used to study the role of cyclic AMP (cAMP), and its downstream mediators. Expression of VEGF and HIF‐1α mRNA were measured by quantitative RT‐PCR; VEGF secretion by ELISA; steroid secretion by radioimmunoassay and HIF‐1α protein levels by western blotting. Inhibitors of adenylate cyclase (AC), protein kinase A (PKA), sarcoma kinase (SrcK), phosphoinositide 3‐kinase (PI3K) and MAP kinase kinase (MEK1/2) abolished the T3‐induced increase in VEGF mRNA and protein levels. The same signalling molecules also mediated the increased production of steroids and HIF‐1α protein in response to T3. Therefore, it was concluded that T3 stimulates steroid secretion and HIF‐1α protein in MLTC‐1 cells through the AC‐cAMP‐PKA‐PI3K‐MEK pathway, which in turn stimulate VEGF production. [ABSTRACT FROM AUTHOR]

Summary: Although the soil bacterium Pseudomonas putida KT2440 bears a bona fide adenylate cyclase gene (cyaA), intracellular concentrations of 3′,5′‐cyclic adenosine monophosphate (cAMP) are barely detectable. By using reporter technology and direct quantification of cAMP under various conditions, we show that such low levels of the molecule stem from the stringent regulation of its synthesis, efflux and degradation. Poor production of cAMP was the result of inefficient translation of cyaA mRNA. Moreover, deletion of the cAMP‐phosphodiesterase pde gene led to intracellular accumulation of the cyclic nucleotide, exposing an additional cause of cAMP drain in vivo. But even such low levels of the signal sustained activation of promoters dependent on the cAMP‐receptor protein (CRP). Genetic and biochemical evidence indicated that the phenomenon ultimately rose from the unusual binding parameters of cAMP to CRP. This included an ultratight cAMP‐CrpP. putida affinity (KD of 45.0 ± 3.4 nM) and an atypical 1:1 effector/dimer stoichiometry that obeyed an infrequent anti‐cooperative binding mechanism. It thus seems that keeping the same regulatory parts and their relational logic but changing the interaction parameters enables genetic devices to take over entirely different domains of the functional landscape. [ABSTRACT FROM AUTHOR]

Optogenetic manipulations have transformed neuroscience in recent years. While sophisticated tools now exist for controlling the firing patterns of neurons, it remains challenging to optogenetically define the plasticity state of individual synapses. A variety of synapses in the mammalian brain express presynaptic long‐term potentiation (LTP) upon elevation of presynaptic cyclic adenosine monophosphate (cAMP), but the molecular expression mechanisms as well as the impact of presynaptic LTP on network activity and behavior are not fully understood. In order to establish optogenetic control of presynaptic cAMP levels and thereby presynaptic potentiation, we developed synaptoPAC, a presynaptically targeted version of the photoactivated adenylyl cyclase bPAC. In cultures of hippocampal granule cells of Wistar rats, activation of synaptoPAC with blue light increased action potential‐evoked transmission, an effect not seen in hippocampal cultures of non‐granule cells. In acute brain slices of C57BL/6N mice, synaptoPAC activation immediately triggered a strong presynaptic potentiation at mossy fiber synapses in CA3, but not at Schaffer collateral synapses in CA1. Following light‐triggered potentiation, mossy fiber transmission decreased within 20 min, but remained enhanced still after 30 min. The optogenetic potentiation altered the short‐term plasticity dynamics of release, reminiscent of presynaptic LTP. Our work establishes synaptoPAC as an optogenetic tool that enables acute light‐controlled potentiation of transmitter release at specific synapses in the brain, facilitating studies of the role of presynaptic potentiation in network function and animal behavior in an unprecedented manner. Read the Editorial Highlight for this article on page 270. [ABSTRACT FROM AUTHOR]