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Adenylate cyclase 3 (ADCY3) is a transmembrane protein predominantly expressed in the primary cilia of neurons. It plays a vital role in converting ATP to cAMP, a secondary messenger that regulates various downstream signaling pathways such as carbohydrates and lipids metabolism. Homozygous loss-of-function variants in the ADCY3 gene lead to severe early-onset obesity and insulin resistance whereas gain-of-function variants protect against obesity. To describe a novel pathogenic ADCY3 variant implicated in early-onset obesity and functionally characterize this variant via in vitro and in silico validation, we identified a novel homozygous nonsense variant c.2520C>G, p.Thr840X in the ADCY3 gene using gene panel sequencing in a four-year-old girl. She was born to first-cousin consanguineous parents. The patient presented with severe obesity, and exhibited hepatomegaly and insulin resistance, with other biochemical and hormonal tests being normal. In vitro and in silico functional analyses showed downregulation and impaired activation of the ADCY3 protein. Our findings contribute to existing research that supports the role of ADCY3 in the genetic pathogenesis of early-onset obesity. In vitro and in silico functional characterization of the novel p.Thr840X variant showed impaired enzymatic activity leading to receptor loss of function, consistent with the patient's phenotype. Genetic testing is essential in severe early-onset obesity and early diagnosis could benefit patients with personalized treatment strategies. [ABSTRACT FROM AUTHOR]

Type three secretion system (TTSS)-competent Pseudomonas aeruginosa expressing soluble promiscuous cyclase, exoenzyme Y (ExoY), generates cyclic nucleotides in pulmonary microvascular endothelial cells (PMVECs). Within cells, cyclic nucleotide signals are highly compartmentalized, but these second messengers are also released into the extracellular space. Although agonist stimulation of endogenous adenylyl cyclase (AC) or the presence of ExoY increases cyclic nucleotides, the proportion of the signal that is in the intracellular versus extracellular compartments is unresolved. Furthermore, it is unclear whether P. aeruginosa primary infection or treatment with sterile media supernatants derived from a primary infection alters beta-adrenergic agonist-induced elevations in cAMP in PMVECs. Herein, we determine that PMVECs release cAMP into the extracellular space constitutively, following beta-adrenergic stimulation of endogenous AC, and following infection with P. aeruginosa expressing ExoY. Surprisingly, in PMVECs, only a small proportion of cGMP is detected within the cell at baseline or following P. aeruginosa ExoY infection with a larger proportion of total cGMP being detected extracellularly. Thus, the ability of lung endothelium to generate cyclic nucleotides may be underestimated by examining intracellular cyclic nucleotides alone, since a large portion is delivered into the extracellular compartment. In addition, P. aeruginosa infection or treatment with sterile media supernatants from a primary infection suppresses the beta-adrenergic cAMP response, which is further attenuated by the expression of functional ExoY. These findings reveal an overabundance of extracellular cyclic nucleotides following infection with ExoY expressing TTSS-competent P. aeruginosa. NEW & NOTEWORTHY:P. aeruginosa exoenzyme Y (ExoY) infection increases cyclic nucleotides intracellularly, but an overabundance of cAMP and cGMP is also detected in the extracellular space and reveals a greater capacity of pulmonary endothelial cells to generate cAMP and cGMP. P. aeruginosa infection or treatment with sterile media supernatants derived from a primary infection suppresses the β-adrenergic-induced cAMP response in pulmonary endothelial cells, which is exacerbated by the expression of functional ExoY. [ABSTRACT FROM AUTHOR]

Purpose: Olfactory dysfunction is increasingly common among COVID-19 patients, impacting their well-being. Reports have demonstrated decreased levels of cyclic adenosine monophosphate and cyclic guanosine monophosphate among patients with chronic olfactory dysfunction. A prospective randomized clinical trial was developed to demonstrate the efficacy of an oral forskolin regimen treatment, an adenylyl cyclase activator that raises intracellular levels of cyclic adenosine monophosphate, for the treatment of olfactory dysfunction following COVID-19, compared to placebo regimen. Methods: The study enrolled 285 participants with persistent olfactory dysfunction post COVID-19 infection, randomly assigning them to receive either placebo capsules (n = 120) or oral forskolin capsules (n = 165). Follow-up was conducted to track progress, with 18 participants from the placebo group and 12 from the forskolin group lost during this period. Olfactory function was assessed using the "Sniffin' Sticks" test, measuring threshold, discrimination and identification scores before and after treatment. Results: Subjects administered forskolin capsules demonstrated a significant enhancement in their composite TDI (threshold, discrimination and identification) score, suggesting a notable amelioration in olfactory functionality. Moreover, the discrimination and identification scores notably improved within the forskolin group. Conversely, no significant alterations were observed in the threshold scores. Conclusion: This study suggests that forskolin can contribute potentially to improve chronic olfactory dysfunction post COVID-19. Trial registration: DFM-IRB00012367-23-10-001. [ABSTRACT FROM AUTHOR]

The nanoscale organization of enzymes associated with the dynamics of second messengers is critical for ensuring compartmentation and localization of signaling molecules in cells. Specifically, the spatiotemporal orchestration of cAMP and Ca2+ oscillations is critical for many cellular functions. Previous experimental studies have shown that the formation of nanodomains of A-kinase anchoring protein 79/150 (AKAP150) and adenylyl cyclase 8 (AC8) on the surface of pancreatic MIN6 β cells modulates the phase of Ca2+-cAMP oscillations from out-of-phase to in-phase. In this work, we develop computational models of the Ca2+/cAMP pathway and AKAP/AC nanodomain formation that give rise to the two important predictions: instead of an arbitrary phase difference, the out-of-phase Ca2+/cAMP oscillation reaches Ca2+ trough and cAMP peak simultaneously, which is defined as inversely out-of-phase; the in-phase and inversely out-of-phase oscillations associated with Ca2+-cAMP dynamics on and away from the nanodomains can be explained by an incoherent feedforward loop. Factors such as cellular surface-to-volume ratio, compartment size, and distance between nanodomains do not affect the existence of in-phase or inversely out-of-phase Ca2+/cAMP oscillation, but cellular surface-to-volume ratio and compartment size can affect the time delay for the inversely out-of-phase Ca2+/cAMP oscillation while the distance between two nanodomains does not. Finally, we predict that both the Turing pattern-generated nanodomains and experimentally measured nanodomains demonstrate the existence of in-phase and inversely out-of-phase Ca2+/cAMP oscillation when the AC8 is at a low level, consistent with the behavior of an incoherent feedforward loop. These findings unveil the key circuit motif that governs cAMP and Ca2+ oscillations and advance our understanding of how nanodomains can lead to spatial compartmentation of second messengers. Author summary: Cyclic adenosine monophosphate (cAMP) and Ca2+ are key molecules that relay signals from the cell membrane to downstream molecules. The temporal and spatial distribution of cAMP and Ca2+ within the cell can be regulated by clusters formed by specific molecules. Previous studies have shown that nanodomains of A-kinase anchoring protein 79/150 (AKAP150) and adenylyl cyclase 8 (AC8) on the surface of pancreatic MIN6 β cells modulates Ca2+-cAMP oscillations from non-zero to zero phase difference, i.e., from out-of-phase to in-phase. By developing a computational model of the Ca2+/cAMP pathway, we found that the non-zero phase difference is not an arbitrary value: it makes Ca2+ reach trough and cAMP reach peak simultaneously. We defined this out-of-phase behavior as inversely out-of-phase, and revealed that this behavior and in-phase behavior can be explained by an incoherent feedforward loop. Biophysical properties of cells and nanodomain distributions do not affect the existence of in-phase or inversely out-of-phase behaviors, but may affect the time delay for the inversely out-of-phase behavior. Furthermore, AKAP/AC nanodomains from a Turing pattern or experiments show consistent simulation results with the incoherent feedforward loop. This study improves our understanding of underlying mechanisms of Ca2+/cAMP oscillation, shedding light on controlling Ca2+ and cAMP through nanodomains. [ABSTRACT FROM AUTHOR]

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]

Dopamine transfers information to striatal neurons, and disrupted neurotransmission leads to motor deficits observed in movement disorders. Striatal dopamine converges downstream to Adenylyl Cyclase Type 5 (AC5)-mediated synthesis of cAMP, indicating the essential role of signal transduction in motor physiology. However, the relationship between dopamine decoding and AC5 regulation is unknown. Here, we utilized an unbiased global protein stability screen to identify Potassium Channel Tetramerization Domain 1 (KCTD1) as a key regulator of AC5 level that is mechanistically tied to N-linked glycosylation. We then implemented a CRISPR/SaCas9 approach to eliminate KCTD1 in striatal neurons expressing a Förster resonance energy transfer (FRET)-based cAMP biosensor. 2-photon imaging of striatal neurons in intact circuits uncovered that dopaminergic signaling was substantially compromised in the absence of KCTD1. Finally, knockdown of KCTD1 in genetically defined dorsal striatal neurons significantly altered motor behavior in mice. These results reveal that KCTD1 acts as an essential modifier of dopaminergic signaling by stabilizing striatal AC5. [ABSTRACT FROM AUTHOR]

Theobromine (TBR) is a methylxanthine known for its bronchodilatory and stimulatory effects. This research evaluated the vitality, capacitation patterns, oxidative characteristics, microbial profile and expression of capacitation-associated proteins (CatSper1/2, sodium bicarbonate cotransporter [NBC], protein kinases A [PKA] and C [PKC] and adenylate cyclase 10 [ADCY10]) in cryopreserved bovine spermatozoa (n = 30) in the absence (cryopreserved control [CtrlC]) or presence of different TBR concentrations (12.5, 25, and 50 µM) in egg yolk extender. Fresh ejaculate served as a negative control (CtrlN). Significant post-thaw maintenance of the sperm motility, membrane and DNA integrity and mitochondrial activity (p < 0.001) were recorded following the administration of 25 μM and 50 μM TBR, then compared to CtrlC. All groups supplemented with TBR exhibited a significantly lower percentage of prematurely capacitated spermatozoa (p < 0.001) than CtrlC. Significantly decreased levels of global reactive oxygen species (ROS), hydrogen peroxide and hydroxyl radicals were observed in the presence of 25 μM and 50 μM TBR (p < 0.01). Western blot analysis revealed that supplementation with 50 μM TBR significantly prevented the loss of NBC and ADCY10 (p < 0.01), while all TBR doses stabilized the levels of PKC (p < 0.05 at 50 μM TBR; p < 0.001 at 12.5 μM and 25 μM TBR). In summary, we suggest that TBR is effective in protecting the spermatozoa during the cryopreservation process through its potential to stimulate energy synthesis while preventing ROS overproduction and the loss of proteins involved in the sperm activation process. [ABSTRACT FROM AUTHOR]

The prolonged overstimulation of β-adrenergic receptors (β-ARs), a member of the G protein-coupled receptor (GPCR) family, causes abnormalities in the density and functionality of the receptor and contributes to cardiac dysfunctions, leading to the development and progression of heart diseases, especially heart failure (HF). Despite recent advancements in HF therapy, mortality and morbidity rates continue to be high. Treatment with β-AR antagonists (β-blockers) has improved clinical outcomes and reduced overall hospitalization and mortality rates. However, several barriers in the management of HF remain, providing opportunities to develop new strategies that focus on the functions and signal transduction of β-ARs involved in the pathogenesis of HF. As β-AR can signal through multiple pathways influenced by different receptor subtypes, expression levels, and signaling components such as G proteins, G protein-coupled receptor kinases (GRKs), β-arrestins, and downstream effectors, it presents a complex mechanism that could be targeted in HF management. In this narrative review, we focus on the regulation of β-ARs at the receptor, G protein, and effector loci, as well as their signal transductions in the physiology and pathophysiology of the heart. The discovery of potential ligands for β-AR that activate cardioprotective pathways while limiting off-target signaling is promising for the treatment of HF. However, applying findings from preclinical animal models to human patients faces several challenges, including species differences, the genetic variability of β-ARs, and the complexity and heterogeneity of humans. In this review, we also summarize recent updates and future research on the regulation of β-ARs in the molecular basis of HF and highlight potential therapeutic strategies for HF. [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Excessive stimulation of the inositol (1,4,5)-trisphosphate (IP3) signaling pathway has been linked to AF through abnormal calcium handling. However, little is known about the mechanisms involved in this process. We expressed the fluorescence resonance energy transfer (FRET)-based cytosolic cyclic adenosine monophosphate (cAMP) sensor EPAC-SH187 in neonatal rat atrial myocytes (NRAMs) and neonatal rat ventricular myocytes (NRVMs). In NRAMs, the addition of the α1-agonist, phenylephrine (PE, 3 µM), resulted in a FRET change of 21.20 ± 7.43%, and the addition of membrane-permeant IP3 derivative 2,3,6-tri-O-butyryl-myo-IP3(1,4,5)-hexakis(acetoxymethyl)ester (IP3-AM, 20 μM) resulted in a peak of 20.31 ± 6.74%. These FRET changes imply an increase in cAMP. Prior application of IP3 receptor (IP3R) inhibitors 2-aminoethyl diphenylborinate (2-APB, 2.5 μM) or Xestospongin-C (0.3 μM) significantly inhibited the change in FRET in NRAMs in response to PE. Xestospongin-C (0.3 μM) significantly inhibited the change in FRET in NRAMs in response to IP3-AM. The FRET change in response to PE in NRVMs was not inhibited by 2-APB or Xestospongin-C. Finally, the localization of cAMP signals was tested by expressing the FRET-based cAMP sensor, AKAP79-CUTie, which targets the intracellular surface of the plasmalemma. We found in NRAMs that PE led to FRET change corresponding to an increase in cAMP that was inhibited by 2-APB and Xestospongin-C. These data support further investigation of the proarrhythmic nature and components of IP3-induced cAMP signaling to identify potential pharmacological targets. NEW & NOTEWORTHY: This study shows that indirect activation of the IP3 pathway in atrial myocytes using phenylephrine and direct activation using IP3-AM leads to an increase in cAMP and is in part localized to the cell membrane. These changes can be pharmacologically inhibited using IP3R inhibitors. However, the cAMP rise in ventricular myocytes is independent of IP3R calcium release. Our data support further investigation into the proarrhythmic nature of IP3-induced cAMP signaling. [ABSTRACT FROM AUTHOR]

Hippocampal neurons exhibit activation of both the conventional transmembrane adenylyl cyclases (tmACs) and the non-canonical soluble adenylyl cyclase (sAC) as sources of cyclic AMP (cAMP). These two cAMP sources play crucial roles in mediating signaling pathways downstream of CRHR1 in neuronal and neuroendocrine contexts. In this study, we investigate the involvement of both cAMP sources in the molecular mechanisms triggered by CRHR2α. Here we provide evidence demonstrating that UCN1 and UCN3 exert a neuritogenic effect on HT22-CRHR2α cells, which is solely dependent on the cAMP pool generated by sAC and PKA activity but independent of ERK1/2 activation. Through the characterization of the effectors implicated in neurite elongation, we found that CREB phosphorylation and c-Fos induction rely on PKA activity and ERK1/2 phosphorylation, underscoring the critical role of signaling pathway regulation. These findings strengthen the concept that localized cAMP microdomains actively participate in the regulation of these signaling processes. [ABSTRACT FROM AUTHOR]

Variants of the G protein–coupled receptor 75 (GPR75) are associated with a lower BMI in large-scale human exome- sequencing studies. However, how GPR75 regulates body weight remains poorly understood. Using random germline mutagenesis in mice, we identified a missense allele (Thinner) of Gpr75 that resulted in a lean phenotype and verified the decreased body weight and fat weight in Gpr75-knockout (Gpr75–/–) mice. Gpr75–/– mice displayed reduced food intake under high-fat diet (HFD) feeding, and pair-feeding normalized their body weight. The endogenous GPR75 protein was exclusively expressed in the brains of 3xFlag-tagged Gpr75-knockin (3xFlag-Gpr75) mice, with consistent expression across different brain regions. GPR75 interacted with Gαq to activate various signaling pathways after HFD feeding. Additionally, GPR75 was localized in the primary cilia of hypothalamic cells, whereas the Thinner mutation (L144P) and human GPR75 variants in individuals with a lower BMI failed to localize in the cilia. Loss of GPR75 selectively inhibited weight gain in HFD-fed mice but failed to suppress the development of obesity in leptin ob–mutant (Lepob-mutant) mice and adenylate cyclase 3–mutant (Adcy3-mutant) mice on a chow diet. Our data reveal that GPR75 is a ciliary protein expressed in the brain and plays an important role in regulating food intake. [ABSTRACT FROM AUTHOR]

Overgrowth of Gardnerella vaginalis causes an imbalance in vaginal microecology. The pathogenicity of G. vaginalis is directly regulated by the cAMP receptor protein (CRP). In this study, we resolve the crystal structure of CRPGv at a resolution of 2.22 Å and find some significant differences from homologous proteins. The first 23 amino acids of CRPGv are inserted into the ligand binding pocket, creating a strong steric barrier to ligand entry that has not been seen previously in its homologues. In the absence of ligands, the two α helices used by CRPGv to bind oligonucleotide chains are exposed and can specifically bind TGTGA-N6-TCACA sequences. cAMP and other ligands of CRP homologs are not cofactors of CRPGv. There is no coding gene of the adenylate cyclase, and cAMP could not be identified in G. vaginalis by liquid chromatography tandem mass spectrometry. We speculate that CRPGv may achieve fine regulation through a conformational transformation different from that of its homologous proteins, and this conformational transformation is no longer dependent on small molecules, but may be aided by accessory proteins. CRPGv is the first discovered CRP that is not ligand-regulated, and its active conformation provides a structural basis for drug screening. The insertion of N-terminal amino acids into the ligand binding pocket prevents the cAMP receptor protein of Gardnerella vaginalis from binding to small molecules [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]

Forskolin (FSK) is a potent adenylate cyclase activator and may display endocrine-disruptive effects via the disruption of steroidogenesis. Here, we tested this hypothesis by use of the in vitro H295R steroidogenesis assay and the in vivo long-term medaka (Oryzias latipes) exposure assay. The results from the H295R assay demonstrated that the transcriptional levels of a series of genes involved in steroidogenesis, including HSD3B2, CYP11A, CYP11B2, CYP17, CYP19, and CYP21, were remarkably up-regulated. Meanwhile, the productions of estrogens (17β-estradiol (17β-E2) and estrone (E1)) and progestins (progesterone (PGT) and 17-hydroxyprogesterone (17-HPT)) were significantly increased, and those of androgens (androstenedione (ADD) and testosterone (TTR)) were significantly inhibited. After waterborne exposure of medaka to FSK for 100 days, the gene expressions of HMGR, HSD17B1, CYP17B, CYP19A, and CYP21A were significantly enhanced in the gonads of male medaka. 17β-E2 was remarkably induced, although without statistical significance. In addition, the biomarker genes for estrogenicity, including VTG-I, VTG-II, CHG-H, and CHG-L, were significantly induced in male medaka livers. Pathological damage to their gonads was further identified. Therefore, the results demonstrated that FSK modulates the transcriptions of steroidogenesis genes and alters hormone levels in vitro and in vivo, which is a mark of endocrine disruption in organisms. [ABSTRACT FROM AUTHOR]

Mice with cardiac-specific overexpression of adenylyl cyclase (AC) type 8 (TGAC8) are under a constant state of severe myocardial stress. They have a remarkable ability to adapt to this stress, but they eventually develop accelerated cardiac aging and experience reduced longevity. We have previously demonstrated through bioinformatics that constitutive adenylyl cyclase activation in TGAC8 mice is associated with the activation of inflammation-related signaling pathways. However, the immune response associated with chronic myocardial stress in the TGAC8 mouse remains unexplored. Here we demonstrate that chronic activation of adenylyl cyclase in cardiomyocytes of TGAC8 mice results in activation of cell-autonomous RelA-mediated NF-κB signaling. This is associated with non-cell-autonomous activation of proinflammatory and age-associated signaling in myocardial endothelial cells and myocardial smooth muscle cells, expansion of myocardial immune cells, increase in serum levels of inflammatory cytokines, and changes in the size or composition of lymphoid organs. All these changes precede the appearance of cardiac fibrosis. We provide evidence indicating that RelA activation in cardiomyocytes with chronic activation of adenylyl cyclase is mediated by calcium-protein Kinase A (PKA) signaling. Using a model of chronic cardiomyocyte stress and accelerated aging, we highlight a novel, calcium/PKA/RelA-dependent connection between cardiomyocyte stress, myocardial inflammation, and systemic inflammation. These findings suggest that RelA-mediated signaling in cardiomyocytes might be an adaptive response to stress that, when chronically activated, ultimately contributes to both cardiac and systemic aging. [ABSTRACT FROM AUTHOR]

The article titled "Slow and rapid auxin responses in Arabidopsis" explores the intricate nature of auxin signaling in plants. It explains the well-known TIR1/AFB–Aux/IAA–ARF pathway that regulates transcriptional regulation and developmental processes associated with auxin. However, recent studies have uncovered rapid auxin responses that occur within seconds. The article provides an overview of the signaling components involved in both slow and rapid auxin responses, emphasizing the complexity of auxin signaling in plants. It discusses the roles of different proteins in auxin signaling and their involvement in various plant developmental processes. The article also mentions the potential contributions of cAMP and cGMP as second messengers in auxin signaling. Additionally, it highlights the ABP1-ABL1/2-TMK cell surface receptor complex's role in slow auxin responses and the involvement of RAF-like kinases in rapid auxin-induced protein phosphorylation. The article concludes by emphasizing the need for further research to fully understand the integration of slow and rapid auxin responses and the evolution of auxin signaling pathways. [Extracted from the article]

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]

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Acinetobacter baumannii is an opportunistic nosocomial pathogen with high morbidity and mortality rates. Current treatment options for this pathogen are limited due to its increasing resistance to last-resort antibiotics. Despite A. baumannii's leading position in the World Health Organisations priority pathogens list, little is known about its virulence regulation. Through a high-throughput screening approach to identify novel biofilm regulators, we identified a previously uncharacterised predicted adenylate cyclase (AC), CavA, as a central regulator of this phenotype. cAMP is a crucial mediator of various aspects of bacterial physiology in other species but information about its role in A. baumannii is limited. We confirm that CavA AC is functional and synthesizes cAMP in A. baumannii. Using dRNA-seq, we verify that CavA is a negative biofilm formation regulator affecting Csu pili and exopolysaccharide production. We demonstrate for the first time that in A. baumannii, cAMP is atop of a hierarchical signalling cascade controlling inter- and intrabacterial signalling by modulating quorum sensing and cyclic di-GMP systems, ultimately governing virulence in vivo and adaptive antibiotic resistance. In contrast to the well-established paradigm in other bacteria where cAMP and cyclic di-GMP levels are inversely regulated, we uncover that the levels of these second messengers are directly proportional in A. baumannii. Overall, this study uncovers the central role of CavA and cAMP in the pathogenic success of A. baumannii and highlights this signalling cascade as a high potential target for novel therapeutic development. Author summary: Acinetobacter baumannii is at the top of the World Health Organisation's list of critical pathogens in urgent need of novel therapeutic interventions as currently treatments options routinely fail due to its high resistance to antibiotics. Despite the importance of this pathogen, we have limited knowledge of how A. baumannii regulates virulence or antimicrobial susceptibility. In this study we show that CavA, an enzyme responsible for the production of the second messenger molecule cyclic AMP (cAMP), decreases biofilm formation while making the bacterium more motile. We demonstrate for the first time in this pathogen, that cAMP controls other signalling systems such as quorum sensing. Also, cAMP increases the levels of another important second messenger molecule cyclic di-GMP, which does not follow the current dogma established in other bacteria. Finally, we show that cAMP increases A. baumannii antibiotic resistance and virulence and provide evidence that cAMP controls many of these phenotypes by binding to the cAMP effector protein Vfr. Altogether, this study presents the important role of cAMP signalling in the success of A. baumannii as human pathogen. This will help develop new therapeutics for better treatment of infections caused by this critical-priority bacterium. [ABSTRACT FROM AUTHOR]

A long-held tenet in inositol-lipid signaling is that cleavage of membrane phosphoinositides by phospholipase Cβ (PLCβ) isozymes to increase cytosolic Ca2+ in living cells is exclusive to Gq- and Gi-sensitive G protein-coupled receptors (GPCRs). Here we extend this central tenet and show that Gs-GPCRs also partake in inositol-lipid signaling and thereby increase cytosolic Ca2+. By combining CRISPR/Cas9 genome editing to delete Gαs, the adenylyl cyclase isoforms 3 and 6, or the PLCβ1-4 isozymes, with pharmacological and genetic inhibition of Gq and G11, we pin down Gs-derived Gβγ as driver of a PLCβ2/3-mediated cytosolic Ca2+ release module. This module does not require but crosstalks with Gαs-dependent cAMP, demands Gαq to release PLCβ3 autoinhibition, but becomes Gq-independent with mutational disruption of the PLCβ3 autoinhibited state. Our findings uncover the key steps of a previously unappreciated mechanism utilized by mammalian cells to finetune their calcium signaling regulation through Gs-GPCRs. Gs heterotrimers are considered to be poor providers of free Gβγ subunits. Here, the authors show that—despite this—Gs-derived Gβγ dimers are active transducers of GPCR-initiated Ca2+ signals involving phosphoinositide-based signaling routes. [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]

Migraine is a complex neurological disorder characterized by recurrent headaches accompanied by sensory disturbances. It involves a combination of genetic, environmental, and neurovascular factors. The objective of this systematic review and meta-analysis was to investigate the correlation between migraine and specific mutant genes by examining the association of Pituitary Adenylate Cyclase-Activating Peptide-38 genes with migraine. A comprehensive search was conducted in major scientific databases, such as PubMed, Scopus, and Embase, to identify relevant studies published up until September 2023. The inclusion criteria encompassed studies that examined the genes (PACAP-38) and various aspects of migraine. Two independent reviewers performed data extraction and quality assessment to ensure the accuracy and reliability of the collected information. Seven studies, comprising 737 patients, were included in the final analysis. The random effects model yielded a standardized mean difference (SMD) of 0.55 (95% CI:-0.15 to 1.25, t=1.45, p=0.19). Heterogeneity among the studies was substantial, with I² indicating 93% variability (95% CI: 84.6% to 96.5%). The heterogeneity was statistically significant (Q= 87.2, df=6, p<0.001). The prediction interval ranged from -1.40 to 2.51. This systematic review and meta-analysis establish a strong link between PACAP-38 and susceptibility to migraine. These findings highlight the significance of genetic factors in migraine development, emphasizing the need for further investigation to elucidate underlying mechanisms and explore the clinical implications of these genetic associations. Keywords: Meta-analysis, migraine, mutant gene, review. [ABSTRACT FROM AUTHOR]

Lung cancer has the highest fatality rate among malignant tumors in the world. Finding new biomarkers of drug resistance is of great importance in the prognosis of lung cancer patients. We found that the polymorphisms of Adenylate Cyclase 1 (ADCY1) are significantly associated with platinum-based chemotherapy resistance in lung cancer patients in our previous research. In this study, we wanted to identify the mechanism of ADCY1 affecting platinum resistance. We used an MTT assay to find if the expression of ADCY1 is associated with the sensitivity of cisplatin in A549, H1299, and A549-DDP cells. Then, we performed CCK-8 tests to detect the absorbance of these cells stimulated by ADCY1, which can discover the cell proliferation that is affected by ADCY1. We investigated cell apoptosis and cell cycles regulated by ADCY1 through the flow cytometry assay. RNA sequencing was used to find the downstream genes affected by ADCY1 which may be associated with drug resistance in lung cancer patients. ADCY1 has higher expression in lung cancer cells than in normal cells. ADCY1 can affect cisplatin resistance in lung cancer cells by regulating cell proliferation, cell apoptosis, and the cell cycle. It may control cell apoptosis by regulating the classical apoptosis biomarkers Bax and Bcl2. Our study showed that ADCY1 may be a new biomarker in the prognosis of lung cancer patients. Much work remains to be carried out to clarify the mechanism in this important emerging field. [ABSTRACT FROM AUTHOR]

Increasing numbers of reports have revealed novel catalytically active cryptic guanylate cyclases (GCs) and adenylate cyclases (ACs) operating within complex proteins in prokaryotes and eukaryotes. Here we review the structural and functional aspects of some of these cyclases and provide examples that illustrate their roles in the regulation of the intramolecular functions of complex proteins, such as the phytosulfokine receptor (PSKR), and reassess their contribution to signal generation and tuning. Another multidomain protein, Arabidopsis thaliana K+ uptake permease (AtKUP5), also harbors multiple catalytically active sites including an N-terminal AC and C-terminal phosphodiesterase (PDE) with an abscisic acid-binding site. We argue that this architecture may enable the fine-tuning and/or sensing of K+ flux and integrate hormone responses to cAMP homeostasis. We also discuss how searches with motifs based on conserved amino acids in catalytic centers led to the discovery of GCs and ACs and propose how this approach can be applied to discover hitherto masked active sites in bacterial, fungal, and animal proteomes. Finally, we show that motif searches are a promising approach to discover ancient biological functions such as hormone or gas binding. [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]

Most neurons are not replaced after injury and thus possess robust intrinsic mechanisms for repair after damage. Axon injury triggers a calcium wave, and calcium and cAMP can augment axon regeneration. In comparison to axon regeneration, dendrite regeneration is poorly understood. To test whether calcium and cAMP might also be involved in dendrite injury signaling, we tracked the responses of Drosophila dendritic arborization neurons to laser severing of axons and dendrites. We found that calcium and subsequently cAMP accumulate in the cell body after both dendrite and axon injury. Two voltage-gated calcium channels (VGCCs), L-Type and T-Type, are required for the calcium influx in response to dendrite injury and play a role in rapid initiation of dendrite regeneration. The AC8 family adenylyl cyclase, Ac78C, is required for cAMP production after dendrite injury and timely initiation of regeneration. Injury-induced cAMP production is sensitive to VGCC reduction, placing calcium upstream of cAMP generation. We propose that two VGCCs initiate global calcium influx in response to dendrite injury followed by production of cAMP by Ac78C. This signaling pathway promotes timely initiation of dendrite regrowth several hours after dendrite damage. Author summary: To last a lifetime, neurons must have robust stress and injury response pathways. While axon regeneration has long been appreciated as an important neuronal repair pathway, dendrite regeneration has only recently emerged as a potential contributor to neuronal health. The first step in mounting an injury response is detecting the injury. We ask whether axonal injury signals calcium and cAMP also play a role in sensing dendrite injury. We show that, although axon and dendrite injury trigger different growth pathways, they both elicit similar global calcium and cAMP elevation in the minute following injury. We identify the proteins responsible for this early calcium and cAMP rise in response to dendrite injury and show that they are important for rapid initiation of dendrite outgrowth. This characterization of early events in the dendrite injury response is an important foundation for understanding how dendrite regeneration works and how different responses are triggered by axon and dendrite injury. [ABSTRACT FROM AUTHOR]

Insig-1 and Insig-2 are endoplasmic reticulum (ER) proteins that inhibit lipid synthesis by blocking transport of sterol regulatory element-binding proteins (SREBP-1 and SREBP-2) from ER to Golgi. In the Golgi, SREBPs are processed proteolytically to release their transcription-activating domains, which enhance the synthesis of fatty acids, triglycerides, and cholesterol. Heretofore, the two Insigs have redundant functions, and there is no rationale for two isoforms. The current data identify a specific function for Insig-2. We show that eicosapentaenoic acid (EPA), a polyunsaturated fatty acid, inhibits fatty acid synthesis in human fibroblasts and rat hepatocytes by activating adenylate cyclase, which induces protein kinase A (PKA) to phosphorylate serine-106 in Insig-2. Phosphorylated Insig-2 inhibits the proteolytic processing of SREBP-1, thereby blocking fatty acid synthesis. Phosphorylated Insig-2 does not block the processing of SREBP-2, which activates cholesterol synthesis. Insig-1 lacks serine-106 and is not phosphorylated at this site. EPA inhibition of SREBP-1 processing was reduced by the replacement of serine-106 in Insig-2 with alanine or by treatment with KT5720, a PKA inhibitor. Inhibition did not occur in mutant human fibroblasts that possess Insig-1 but lack Insig-2. These data provide an Insig-2- specific mechanism for the long-known inhibition of fatty acid synthesis by polyunsaturated fatty acids. [ABSTRACT FROM AUTHOR]

Over half of spinal cord injury (SCI) patients develop opioid-resistant chronic neuropathic pain. Safer alternatives to opioids for treatment of neuropathic pain are gabapentinoids (e.g., pregabalin and gabapentin). Clinically, gabapentinoids appear to amplify opioid effects, increasing analgesia and overdose-related adverse outcomes, but in vitro proof of this amplification and its mechanism are lacking. We previously showed that after SCI, sensitivity to opioids is reduced by fourfold to sixfold in rat sensory neurons. Here, we demonstrate that after injury, gabapentinoids restore normal sensitivity of opioid inhibition of cyclic AMP (cAMP) generation, while reducing nociceptor hyperexcitability by inhibiting voltage-gated calcium channels (VGCCs). Increasing intracellular Ca2+ or activation of L-type VGCCs (L-VGCCs) suffices to mimic SCI effects on opioid sensitivity, in a manner dependent on the activity of the Raf1 proto-oncogene, serine/threonine-protein kinase C-Raf, but independent of neuronal depolarization. Together, our results provide a mechanism for potentiation of opioid effects by gabapentinoids after injury, via reduction of calcium influx through L-VGCCs, and suggest that other inhibitors targeting these channels may similarly enhance opioid treatment of neuropathic pain. [ABSTRACT FROM AUTHOR]

Background: Intracellular sensing of lipopolysaccharide (LPS) is essential for the immune response against gram-negative bacteria and results in activation of caspase-11 and pyroptotic cell death with fatal consequences in sepsis. We found the neuronal guidance receptor plexin C1 (PLXNC1) influences the intracellular response to LPS. Methods: We employed a murine model of sepsis via cecal ligation and binding (CLP), using PLXNC1-/- mice and littermate controls, and additionally transfected murine bone-marrow-derived macrophages (BMDMs) from both genotypes with LPS to achieve activation of the noncanonical inflammasome ex vivo. Additionally, we transfected the PLXNC1 ligand SL4c-d in vivo and ex vivo to examine its effect on intracellular LPS response. Results: We found the neuronal guidance receptor PLXNC1 dampens the intracellular response to LPS by interacting with adenylate cyclase 4 (ADCY4) and protein kinase A activity, which in turn diminishes caspase-11 expression. The absence of PLXNC1 results in excessive inflammation marked by increased cytokine release, increased secondary organ injury and reduced sepsis survival in a murine sepsis model induced by CLP. Notably, administration of SL4c-d—peptide ligand of PLXNC1—reduces the inflammatory response during CLP-induced sepsis and improves survival. Conclusions: These results elucidate a previously unknown mechanism for PLXNC1 suppressing excessive noncanonical inflammasome activity and offer a new potential target for treatment of sepsis with its detrimental effects. [ABSTRACT FROM AUTHOR]

Introduction: Air pollution from diesel combustion is linked in part to the generation of diesel exhaust particles (DEP). DEP exposure induces various processes, including inflammation and oxidative stress, which ultimately contribute to a decline in lung function. Cyclic AMP (cAMP) signaling is critical for lung homeostasis. The impact of DEP on cAMP signaling is largely unknown. Methods: We exposed human bronchial epithelial (BEAS-2B) cells to DEP for 24-72 h and evaluated mitochondrial bioenergetics, markers of oxidative stress and inflammation and the components of cAMP signaling. Mitochondrial bioenergetics was measured at 72 h to capture the potential and accumulative effects of prolonged DEP exposure on mitochondrial function. Results: DEP profoundly altered mitochondrial morphology and network integrity, reduced both basal and ATP-linked respiration as well as the glycolytic capacity of mitochondria. DEP exposure increased gene expression of oxidative stress and inflammation markers such as interleukin-8 and interleukin-6. DEP significantly affected mRNA levels of exchange protein directly activated by cAMP-1 and -2 (Epac1, Epac2), appeared to increase Epac1 protein, but left phospho-PKA levels unhanged. DEP exposure increased A-kinase anchoring protein 1, β2-adrenoceptor and prostanoid E receptor subtype 4 mRNA levels. Interestingly, DEP decreased mRNA levels of adenylyl cyclase 9 and reduced cAMP levels stimulated by forskolin (AC activator), fenoterol (β2-AR agonist) or PGE2 (EPR agonist). Discussion: Our findings suggest that DEP induces mitochondrial dysfunction, a process accompanied by oxidative stress and inflammation, and broadly dampens cAMP signaling. These epithelial responses may contribute to lung dysfunction induced by air pollution exposure. [ABSTRACT FROM AUTHOR]

The intestinal barrier plays a crucial role in homeostasis by both facilitating the absorption of nutrients and fluids and providing a tight shield to prevent the invasion by either pathogen or commensal microorganisms. Intestinal barrier malfunction is associated with systemic inflammation, oxidative stress, and decreased insulin sensitivity, which may lead to the dysregulation of other tissues. Therefore, a deeper understanding of physiological aspects related to an enhanced barrier function is of significant scientific and clinical relevance. The naked mole-rat has many unusual biological features, including attenuated colonic neuron sensitivity to acid and bradykinin and resistance to chemical-induced intestinal damage. However, insight into their intestinal barrier physiology is scarce. Here, we observed notable macroscopic and microscopic differences in intestinal tissue structure between naked mole-rats and mice. Moreover, naked mole-rats showed increased number of larger goblet cells and elevated mucus content. In measuring gut permeability, naked mole-rats showed reduced permeability compared with mice, measured as transepithelial electrical resistance, especially in ileum. Furthermore, intestinal ion secretion induced by serotonin, bradykinin, histamine, and capsaicin was significantly reduced in naked mole-rats compared with mice, despite the expression of receptors for all these agonists. In addition, naked mole-rats exhibited reduced prosecretory responses to the nonselective adenylate cyclase activator forskolin. Collectively, these findings indicate that naked mole-rats possess a robust and hard-to-penetrate gastrointestinal barrier that is resistant to environmental and endogenous irritants. Naked mole-rats may therefore provide valuable insights into the physiology of the intestinal barrier and set the stage for the development of innovative and effective therapies. NEW & NOTEWORTHY This is the first study to characterize the intestinal function of naked mole-rats. We found that these animals show a robust gut tissue structure, displaying thicker intestinal layers, longer villi, and larger crypts. Naked mole-rats showed more and larger goblet cells, with increased mucus content. Intestinal permeability, especially in the ileum, was substantially lower than that of mice. Finally, naked mole-rats showed reduced intestinal anion secretion in response to serotonin, bradykinin, histamine, capsaicin, and forskolin. [ABSTRACT FROM AUTHOR]

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent Cl− channel, is closely associated with multiple pathogen infections, such as SARS-CoV-2. However, whether the function of the CFTR is involved in herpes simplex virus (HSV) infection has not been reported. To evaluate the association of CFTR activity with HSV infection, the antiviral effect of CFTR inhibitors in epithelial cells and HSV-infected mice was tested in this study. The data showed that treatment with CFTR inhibitors in different concentrations, Glyh-101 (5–20 μM), CFTRi-172 (5–20 μM) and IOWH-032 (5–20 μM), or the gene silence of the CFTR could suppress herpes simplex virus 1 (HSV-1) and herpes simplex virus 2 (HSV-2) replication in human HaCaT keratinocytes cells, and that a CFTR inhibitor, Glyh-101 (10–20 μM), protected mice from HSV-1 and HSV-2 infection. Intracellular Cl− concentration ([Cl−]i) was decreased after HSV infection via the activation of adenylyl cyclase (AC)-cAMP signaling pathways. CFTR inhibitors (20 μM) increased the reduced [Cl−]i caused by HSV infection in host epithelial cells. Additionally, CFTR inhibitors reduced the activity and phosphorylation of SGK1 in infected cells and tissues (from the eye and vagina). Our study found that CFTR inhibitors can effectively suppress HSV-1 and HSV-2 infection, revealing a previously unknown role of CFTR inhibitors in HSV infection and suggesting new perspectives on the mechanisms governing HSV infection in host epithelial cells, as well as leading to potential novel treatments. [ABSTRACT FROM AUTHOR]

The vagal regulation of cardiac function involves acetylcholine (ACh) receptor activation followed by negative chronotropic and negative as well as positive inotropic effects. The resulting signaling pathways may include Gi/o protein-coupled reduction in adenylyl cyclase (AC) activity, direct Gi/o protein-coupled activation of ACh-activated potassium current (IKACh), inhibition of L-type calcium ion channels, and/or the activation of protein phosphatases. Here, we studied the role of the protein phosphatases 1 (PP1) and 2A (PP2A) for muscarinic receptor signaling in isolated atrial preparations of transgenic mice with cardiomyocyte-specific overexpression of either the catalytic subunit of PP2A (PP2A-TG) or the inhibitor-2 (I2) of PP1 (I2-TG) or in double transgenic mice overexpressing both PP2A and I2 (DT). In mouse left atrial preparations, carbachol (CCh), cumulatively applied (1 nM–10 µM), exerted at low concentrations a negative inotropic effect followed by a positive inotropic effect at higher concentrations. This biphasic effect was noted with CCh alone as well as when CCh was added after β-adrenergic pre-stimulation with isoprenaline (1 µM). Whereas the response to stimulation of β-adrenoceptors or adenosine receptors (used as controls) was changed in PP2A-TG, the response to CCh was unaffected in atrial preparations from all transgenic models studied here. Therefore, the present data tentatively indicate that neither PP2A nor PP1, but possibly other protein phosphatases, is involved in the muscarinic receptor-induced inotropic and chronotropic effects in the mouse heart. [ABSTRACT FROM AUTHOR]

Photoregulated adenylate cyclases bPAC and OaPAC are compared using methods of classical molecular dynamics in the states before and after irradiation with light. The pathways of signal transduction from the photoreceptor domain to the catalytic domain are determined. The more pronounced acceleration of the catalytic reaction as a result of irradiation with light in the case of bPAC is determined both by a shift of the equilibrium towards a closed conformation and a more significant increase in the number of suboptimal pathways of allosteric signal transduction from one domain to another. [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]

Synaptic plasticity is a key cellular model for learning, memory and chronic pain. Most previous studies were carried out in rats and mice, and less is known about synaptic plasticity in non-human primates. In the present study, we used integrative experimental approaches to study long-term potentiation (LTP) in the anterior cingulate cortex (ACC) of adult tree shrews. We found that glutamate is the major excitatory transmitter and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionicacid (AMPA) receptors mediate postsynaptic responses. LTP in tree shrews was greater than that in adult mice and lasted for at least 5 h. N-methyl-d-aspartic acid (NMDA) receptors, Ca2+ influx and adenylyl cyclase 1 (AC1) contributed to tree shrew LTP. Our results suggest that LTP is a major form of synaptic plasticity in the ACC of primate-like animals. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'. [ABSTRACT FROM AUTHOR]

GABAergic transmission is influenced by post-translational modifications, like phosphorylation, impacting channel conductance, allosteric modulator sensitivity, and membrane trafficking. O-GlcNAcylation is a post-translational modification involving the O-linked attachment of β-N-acetylglucosamine on serine/threonine residues. Previously we reported an acute increase in O-GlcNAcylation elicits a long-term depression of evoked GABAAR inhibitory postsynaptic currents (eIPSCs) onto hippocampal principal cells. Importantly, O-GlcNAcylation and phosphorylation can co-occur or compete for the same residue; whether they interact in modulating GABAergic IPSCs is unknown. We tested this by recording IPSCs from hippocampal principal cells and pharmacologically increased O-GlcNAcylation, before or after increasing serine phosphorylation using the adenylate cyclase activator, forskolin. Although forskolin had no significant effect on baseline eIPSC amplitude, we found that a prior increase in O-GlcNAcylation unmasks a forskolin-dependent increase in eIPSC amplitude, reversing the O-GlcNAc-induced eIPSC depression. Inhibition of adenylate cyclase or protein kinase A did not prevent the potentiating effect of forskolin, indicating serine phosphorylation is not the mechanism. Surprisingly, increasing O-GlcNAcylation also unmasked a potentiating effect of the neurosteroids 5α-pregnane-3α,21-diol-20-one (THDOC) and progesterone on eIPSC amplitude in about half of the recorded cells, mimicking forskolin. Our findings show that under conditions of heightened O-GlcNAcylation, the neurosteroid site on synaptic GABAARs is possibly accessible to agonists, permitting strengthening of synaptic inhibition. [ABSTRACT FROM AUTHOR]

Pain and anxiety are two common and undertreated non-motor symptoms in Parkinson's disease (PD), which affect the life quality of PD patients, and the underlying mechanisms remain unclear. As an important subtype of adenylyl cyclases (ACs), adenylyl cyclase subtype 1 (AC1) is critical for the induction of cortical long-term potentiation (LTP) and injury induced synaptic potentiation in the cortical areas including anterior cingulate cortex (ACC) and insular cortex (IC). Genetic deletion of AC1 or pharmacological inhibition of AC1 improved chronic pain and anxiety in different animal models. In this study, we proved the motor deficit, pain and anxiety symptoms of PD in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice model. As a lead candidate AC1 inhibitor, oral administration (1 dose and seven doses) of NB001 (20 and 40 mg/kg) showed significant analgesic effect in MPTP-treated mice, and the anxiety behavior was also reduced (40 mg/kg). By using genetic knockout mice, we found that AC1 knockout mice showed reduced pain and anxiety symptoms after MPTP administration, but not AC8 knockout mice. In summary, genetic deletion of AC1 or pharmacological inhibition of AC1 improved pain and anxiety symptoms in PD model mice, but didn't affect motor function. These results suggest that NB001 is a potential drug for the treatment of pain and anxiety symptoms in PD patients by inhibiting AC1 target. [ABSTRACT FROM AUTHOR]

Introduction: Drotaverine, paracetamol, and peppermint oil are often prescribed for the treatment of gastrointestinal spasm and pain. This study aimed to evaluate the effect of these drugs alone and combined with the well-known antispasmodic hyoscine butylbromide on the human colon. Methods: Colon samples were obtained from macroscopically normal regions of 68 patients undergoing surgery and studied in muscle bath. Drotaverine, paracetamol, and peppermint oil were tested alone and in combination with hyoscine butylbromide on (1) spontaneous contractility induced by isometric stretch (in the presence of 1 µM tetrodotoxin) and (2) contractility induced by 10-5 M carbachol and after (3) electrical field stimulation-induced selective stimulation of excitatory (in the presence of 1 mM Nε-nitro-L-arginine and 10 µM MRS2179) and (4) inhibitory (under non-adrenergic, non-cholinergic conditions) pathways. (5) Drotaverine alone was also tested on cAMPdependent pathway activated by forskolin. Results: Compared with the vehicle, drotaverine and paracetamol (10-9-10-5 M) did not modify spontaneous contractions, carbachol-induced contractions, and responses attributed to selective activation of excitatory pathways. The addition of hyoscine butylbromide (10-7-10-5 M), concentration-dependently reduced myogenic contractions and carbachol- and electrical field stimulation-induced contractile responses. The association of paracetamol (10-4 M) and hyoscine butylbromide (10-7-10-5 M) was not different from hyoscine butylbromide alone (10-7-10-5 M). At higher concentrations (10-3M-3*10-3 M), paracetamol decreased myogenic and carbachol-induced contractions. The adenylate cyclase activator, forskolin, concentration-dependently reduced contractility, leading to smooth muscle relaxation. The effect of forskolin 10-7 M was concentration-dependently enhanced by drotaverine (10-6M-10-5M). Discussion: Peppermint oil reduced myogenic activity and carbachol- and electrical field stimulation-induced contractions. The association of hyoscine butylbromide and peppermint oil was synergistic since the interaction index measured with the isobologram was lower than 1. No effect was seen on the neural-mediated inhibitory responses with any of the drugs studied although peppermint oil reduced the subsequent off-contraction. Drotaverine and hyoscine butylbromide have a complementary effect on human colon motility as one stimulates the cAMP inhibitory pathway and the other inhibits the excitatory pathway. Peppermint oil is synergic with hyoscine butylbromide suggesting that a combination therapy may be more effective in treating patients. In contrast, at therapeutic concentrations, paracetamol does not modify colonic contractility, suggesting that the association of paracetamol and hyoscine butylbromide has independent analgesic and antispasmodic properties. [ABSTRACT FROM AUTHOR]

Heterologous sensitization of adenylyl cyclase (AC) results in elevated cAMP signaling transduction that contributes to drug dependence. Inhibiting cullin3-RING ligases by blocking the neddylation of cullin3 abolishes heterologous sensitization, however, the modulating mechanism remains uncharted. Here, we report an essential role of the potassium channel tetramerization domain (KCTD) protein 2, 5, and 17, especially the dominant isoform KCTD5 in regulating heterologous sensitization of AC1 and morphine dependence via working with cullin3 and the cullin-associated and neddylation-dissociated 1 (CAND1) protein. In cellular models, we observed enhanced association of KCTD5 with Gβ and cullin3, along with elevated dissociation of Gβ from AC1 as well as of CAND1 from cullin3 in heterologous sensitization of AC1. Given binding of CAND1 inhibits the neddylation of cullin3, we further elucidated that the enhanced interaction of KCTD5 with both Gβ and cullin3 promoted the dissociation of CAND1 from cullin3, attenuated the inhibitory effect of CAND1 on cullin3 neddylation, ultimately resulted in heterologous sensitization of AC1. The paraventricular thalamic nucleus (PVT) plays an important role in mediating morphine dependence. Through pharmacological and biochemical approaches, we then demonstrated that KCTD5/cullin3 regulates morphine dependence via modulating heterologous sensitization of AC, likely AC1 in PVT in mice. In summary, the present study revealed the underlying mechanism of heterologous sensitization of AC1 mediated by cullin3 and discovered the role of KCTD proteins in regulating morphine dependence in mice. Heterologous sensitization of adenylyl cyclase (AC) contributes to drug dependence. This study shows that KCTD proteins 2, 5 and 17 regulate AC heterologous sensitization by enabling Cullin3 neddylation, and identifies a crucial role of this pathway in the paraventricular thalamic nucleus in regulating morphine dependence. [ABSTRACT FROM AUTHOR]

Dynamic presynaptic actin remodeling drives structural and functional plasticity at synapses, but the underlying mechanisms remain largely unknown. Previous work has shown that actin regulation via Rac1 guanine exchange factor (GEF) Vav signaling restrains synaptic growth via bone morphogenetic protein (BMP)-induced receptor macropinocytosis and mediates synaptic potentiation via mobilization of reserve pool vesicles in presynaptic boutons. Here, we find that Gef26/PDZ-GEF and small GTPase Rap1 signaling couples the BMP-induced activation of Abelson kinase to this Vav-mediated macropinocytosis. Moreover, we find that adenylate cyclase Rutabaga (Rut) signaling via exchange protein activated by cAMP (Epac) drives the mobilization of reserve pool vesicles during post-tetanic potentiation (PTP). We discover that Rap1 couples activation of RutcAMP- Epac signaling to Vav-mediated synaptic potentiation. These findings indicate that Rap1 acts as an essential, convergent node for Abelson kinase and cAMP signaling to mediate BMP-induced structural plasticity and activity-induced functional plasticity via Vav-dependent regulation of the presynaptic actin cytoskeleton. [ABSTRACT FROM AUTHOR]

Two new binuclear tetranitrosyl iron complexes of the composition [Fe2L2(NO)4] (1, L = 4-methoxybenzenemethanethiolyl; 2, L = 4-chlorobenzenemethanethiolyl) were synthesized. Their crystals were characterized by X-ray diffraction, by IR, ESR, UV, and Mössbauer spectroscopies, and by elemental analysis. The NO-donating properties and transformations of the complexes in physiological solutions, including reactions in the presence of bovine serum albumin, were studied. The cytotoxicity of compounds 1 and 2 was studied on normal Vero cells. The effect of the complexes on enzymes was investigated taking guanylate cyclase and adenylate cyclase as examples. The results obtained allow one to recommend compound 2 for further investigations as a potential drug with vaso- and bronchodilator properties. [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]

Cannabinoids and their receptors play a significant role in the regulation of gastrointestinal (GIT) peristalsis and intestinal barrier permeability. This review critically evaluates current knowledge about the mechanisms of action and biological effects of endocannabinoids and phytocannabinoids on GIT functions and the potential therapeutic applications of these compounds. The results of ex vivo and in vivo preclinical data indicate that cannabinoids can both inhibit and stimulate gut peristalsis, depending on various factors. Endocannabinoids affect peristalsis in a cannabinoid (CB) receptor-specific manner; however, there is also an important interaction between them and the transient receptor potential cation channel subfamily V member 1 (TRPV1) system. Phytocannabinoids such as Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) impact gut motility mainly through the CB1 receptor. They were also found to improve intestinal barrier integrity, mainly through CB1 receptor stimulation but also via protein kinase A (PKA), mitogen-associated protein kinase (MAPK), and adenylyl cyclase signaling pathways, as well as by influencing the expression of tight junction (TJ) proteins. The anti-inflammatory effects of cannabinoids in GIT disorders are postulated to occur by the lowering of inflammatory factors such as myeloperoxidase (MPO) activity and regulation of cytokine levels. In conclusion, there is a prospect of utilizing cannabinoids as components of therapy for GIT disorders. [ABSTRACT FROM AUTHOR]

Simple Summary: Adenylyl cyclase is an enzyme that plays an important role in cellular signalling in mammals. This review gathers current understanding of the role of adenylyl cyclase in the eye, covering the cornea, lens, retina, lacrimal gland and tear film, and explores their potential as targets for therapeutic innovation. Adenylyl cyclases (ACs) are a group of enzymes that convert adenosine-5′-triphosphate (ATP) to cyclic adenosine 3′,5′ monophosphate (cAMP), a vital and ubiquitous signalling molecule in cellular responses to hormones and neurotransmitters. There are nine transmembrane (tmAC) forms, which have been widely studied; however, the tenth, soluble AC (sAC) is less extensively characterised. The eye is one of the most metabolically active sites in the body, where sAC has been found in abundance, making it a target for novel therapeutics and biomarking. In the cornea, AC plays a role in endothelial cell function, which is vital in maintaining stromal dehydration, and therefore, clarity. In the retina, AC has been implicated in axon cell growth and survival. As these cells are irreversibly damaged in glaucoma and injury, this molecule may provide focus for future therapies. Another potential area for glaucoma management is the source of aqueous humour production, the ciliary body, where AC has also been identified. Furthering the understanding of lacrimal gland function is vital in managing dry eye disease, a common and debilitating condition. sAC has been linked to tear production and could serve as a therapeutic target. Overall, ACs are an exciting area of study in ocular health, offering multiple avenues for future medical therapies and diagnostics. This review paper explores the diverse roles of ACs in the eye and their potential as targets for innovative treatments. [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]