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Purpose: Ulcerative colitis (UC) is a chronic, non-specific inflammatory condition of the colon, characterized by recurrent episodes and a notable lack of effective pharmacological treatments. Scutellarin, a natural component, exhibits appreciable pharmacological effects and therapeutic potential for various diseases. However, its effects on UC are not fully understood, and the precise mechanisms remain to be deciphered. This study aimed to assess the therapeutic efficacy of scutellarin and elucidate its underlying mechanisms in treating UC., Methods: This study utilized dextran sulfate sodium (DSS)-induced mice to evaluate the therapeutic potential of scutellarin against UC and to elucidate the mechanisms involving the gut microbiota. An antibiotics cocktail (ABX) and fecal microbiota transplantation (FMT) were also used to determine the mechanistic role of the gut microbiota. An integrative approach combining fecal metabolomics and network pharmacology analysis was used to explore the gut microbiota-directed molecular mechanism., Results: The results showed that scutellarin provided various therapeutic benefits in UC management, including alleviating weight loss, slowing disease progression, and reducing inflammatory damage in colon structures. The improved gut microbiota after scutellarin administration contributed to these effects. Fecal metabolome revealed that scutellarin selectively mitigated DSS-induced dysregulation of gut microbiota-derived metabolites, including glycolic acid, γ-aminobutyric acid, glutamate, tryptophan, xanthine, and β-hydroxypyruvate. Network pharmacology analysis, along with in vivo experimental verification, implicated the cAMP/PKA/NF-κB pathway in the action of these metabolites in treating UC, which may be the mechanism responsible for scutellarin's curative effects on UC., Conclusion: This study demonstrates the potential of scutellarin in alleviating UC by activating the cAMP/PKA/NF-κB pathway through gut microbiota-derived metabolites, highlighting scutellarin as a promising therapeutic agent for UC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

It is widely believed that information storage in neuronal circuits involves nanoscopic structural changes at synapses, resulting in the formation of synaptic engrams. However, direct evidence for this hypothesis is lacking. To test this conjecture, we combined chemical potentiation, functional analysis by paired pre-postsynaptic recordings, and structural analysis by electron microscopy (EM) and freeze-fracture replica labeling (FRL) at the rodent hippocampal mossy fiber synapse, a key synapse in the trisynaptic circuit of the hippocampus. Biophysical analysis of synaptic transmission revealed that forskolin-induced chemical potentiation increased the readily releasable vesicle pool size and vesicular release probability by 146% and 49%, respectively. Structural analysis of mossy fiber synapses by EM and FRL demonstrated an increase in the number of vesicles close to the plasma membrane and the number of clusters of the priming protein Munc13-1, indicating an increase in the number of both docked and primed vesicles. Furthermore, FRL analysis revealed a significant reduction of the distance between Munc13-1 and CaV2.1 Ca2+ channels, suggesting reconfiguration of the channel-vesicle coupling nanotopography. Our results indicate that presynaptic plasticity is associated with structural reorganization of active zones. We propose that changes in potential nanoscopic organization at synaptic vesicle release sites may be correlates of learning and memory at a plastic central synapse., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The phototransduction cascade enables the photoreceptor to detect light over a wide range of intensities without saturation. The main second messenger of the cascade is cGMP and the primary regulatory mechanism is calcium feedback. However, some experimental data suggest that cAMP may also play a role in regulating the phototransduction cascade, but this would require changes in cAMP on a time scale of seconds. Currently, there is a lack of data on the dynamics of changes in intracellular cAMP levels on this timescale. This is largely due to the specificity of the sensory modality of photoreceptors, which makes it practically impossible to use conventional experimental approaches based on fluorescence methods. In this study, we employed the method of rapid cryofixation of retinal samples after light stimulation and subsequent isolation of outer segment preparations. The study employed highly sensitive metabolomics approaches to measure levels of cAMP. Additionally, PKA activity was measured in the samples using a western blot. The results indicate that when exposed to near-saturating but still moderate light, cAMP levels increase transiently within the first second and then return to pre-stimulus levels. The increase in cAMP activates PKA, resulting in the phosphorylation of PKA-specific substrates in frog retinal outer segments., (© 2024 Chernyshkova et al.)

The carcinogenic effects of benzidine (BZ) on bladder cancer are well documented, but its potential for promoting upper urinary tract urothelial carcinoma (UTUC) remains unclear. The ability of emodin, a natural pharmaceutical compound, to prevent BZ‑associated UTUC has not been previously explored. To the best of our knowledge, the present study is the first to reveal that BZ significantly enhanced the survival and migration of UTUC cell lines in vitro . Furthermore, in vivo experiments demonstrated that BZ promoted an increase in the size of subcutaneous tumors in nude mice. Further investigation revealed that BZ upregulated the expression of protein kinase A (PKA) and cyclooxygenase 2 (COX2), along with downstream matrix metalloproteinase 9 (MMP9) and vascular endothelial growth factor (VEGF), in UTUC cells. Moreover, BZ increased the levels of cyclic adenosine monophosphate (cAMP) and prostaglandin E2 (PGE2) in cell lysates. By contrast, emodin reduced the PKA and COX2 expression levels compared with the BZ‑treated group. Similarly, the in vivo experiments demonstrated that emodin significantly inhibited tumor growth in BZ‑pretreated nude mice, accompanied by reductions in the cAMP, PGE2, MMP9 and VEGF levels. These findings elucidated the role of BZ in promoting UTUC progression. Additionally, emodin has emerged as a novel inhibitor of BZ‑induced UTUC development through PKA/COX2 inhibition, suggesting its potential as a natural therapeutic agent against BZ‑associated UTUC.

Metabolic syndrome (MetS) increases the risk of coronary artery disease, but effects of this condition on the working myocardium remain to be fully elucidated. In the present study we evaluated the consequences of diet-induced metabolic disorders on cardiac function and myocyte performance using female mice fed with Western diet. Animals maintained on regular chow were used as control (Ctrl). Mice on the Western diet (WesD) had increased body weight, impaired glucose metabolism, preserved diastolic and systolic function, but increased left ventricular (LV) mass, with respect to Ctrl animals. Moreover, WesD mice had reduced heart rate variability (HRV), indicative of altered cardiac sympathovagal balance. Myocytes from WesD mice had increased volume, enhanced cell mechanics, and faster kinetics of contraction and relaxation. Moreover, levels of cAMP and protein kinase A (PKA) activity were enhanced in WesD myocytes, and interventions aimed at stabilizing cAMP/PKA abrogated functional differences between Ctrl and WesD cells. Interestingly, in vivo β-adrenergic receptor (β-AR) blockade normalized the mechanical properties of WesD myocytes and revealed defective cardiac function in WesD mice, with respect to Ctrl. Collectively, these results indicate that metabolic disorders induced by Western diet enhance the cAMP/PKA signaling pathway, a possible adaptation required to maintain cardiac function., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Ras signaling and glycosylphosphatidylinositol (GPI) biosynthesis are mutually inhibitory in S. cerevisiae (Sc). The inhibition is mediated via an interaction of yeast Ras2 with the Eri1 subunit of its GPI- N -acetylglucosaminyl transferase (GPI-GnT), the enzyme catalyzing the very first GPI biosynthetic step. In contrast, Ras signaling and GPI biosynthesis in C. albicans (Ca) are mutually activated and together control the virulence traits of the human fungal pathogen. What might be the role of Eri1 in this pathogen? The present manuscript addresses this question while simultaneously characterizing the cellular role of CaEri1. It is either nonessential or required at very low levels for cell viability in C. albicans . Severe depletion of CaEri1 results in reduced GPI biosynthesis and cell wall defects. It also produces hyperfilamentation phenotypes in Spider medium as well as in bicarbonate medium containing 5% CO 2 , suggesting that both the Ras-dependent and Ras-independent cAMP-PKA pathways for hyphal morphogenesis are activated in these cells. Pull-down and acceptor-photobleaching FRET experiments suggest that CaEri1 does not directly interact with CaRas1 but does so through CaGpi2, another GPI-GnT subunit. We showed previously that CaGpi2 is downstream of CaEri1 in cross talk with CaRas1 and for Ras-dependent hyphal morphogenesis. Here we show that CaEri1 is downstream of all GPI-GnT subunits in inhibiting Ras-independent filamentation. CaERI1 also participates in intersubunit transcriptional cross talk within the GPI-GnT, a feature unique to C. albicans . Virulence studies using G. mellonella larvae show that a heterozygous strain of CaERI1 is better cleared by the host and is attenuated in virulence.

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., Competing Interests: The authors have declared that no competing interests exist, (Copyright: © 2024 Armando et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Phosphodiesterases (PDEs) are enzymes that catalyze the hydrolysis of cyclic adenosine monophosphate AMP (cAMP) and/or cyclic guanosine monophosphate (cGMP). PDE inhibitors can mitigate chronic pain and depression when these disorders occur individually; however, there is limited understanding of their role in concurrent chronic pain and depression. We aimed to evaluate the mechanisms of action of PDE using 2 mouse models of concurrent chronic pain and depression., Methods: C57BL/6J mice were subjected to partial sciatic nerve ligation (PSNL) to induce chronic neuropathic pain or injected with complete Freund's adjuvant (CFA) to induce inflammatory pain, and both animals showed depression-like behavior. First, we determined the change in PDE expression in both animal models. Next, we determined the effect of PDE7 inhibitor BRL50481 or hippocampal PDE7A knockdown on PSNL- or CFA-induced chronic pain and depression-like behavior. We also investigated the role of cAMP-protein kinase A (PKA)-cAMP response element binding protein (CREB)-brain-derived neurotrophic factor (BDNF) signaling and neuroinflammation in the effect of PDE7A inhibition on PSNL- or CFA-induced chronic pain and depression-like behavior., Results: This induction of chronic pain and depression in the 2 animal models upregulated hippocampal PDE7A. Oral administration of PDE7 inhibitor, BRL50481, or hippocampal PDE7A knockdown significantly reduced mechanical hypersensitivity and depression-like behavior. Hippocampal PDE7 inhibition reversed PSNL- or CFA-induced downregulation of cAMP and BDNF and the phosphorylation of PKA, CREB, and p65. cAMP agonist forskolin reversed these changes and caused milder behavioral symptoms of pain and depression. BRL50481 reversed neuroinflammation in the hippocampus in PSNL mice., Conclusions: Hippocampal PDE7A mediated concurrent chronic pain and depression in both mouse models by inhibiting cAMP-PKA-CREB-BDNF signaling. Inhibiting PDE7A or activating cAMP-PKA-CREB-BDNF signaling are potential strategies to treat concurrent chronic pain and depression., (© The Author(s) 2024. Published by Oxford University Press on behalf of CINP.)

Aging is an inevitable natural process with time-dependent dysfunction and the occurrence of various diseases, which impose heavy burdens on individuals, families, and society. It has been reported that NLRP3 inflammasome-induced pyroptosis contributes significantly to age-related diseases and aging, while TXNIP is suggested to be involved in regulating pyroptosis mediated by NLRP3. However, the mechanism between TXNIP and NLRP3 inflammasome is still unclear. In this study, we used HT-22 cells to explore the effect of TXNIP on pyroptosis and its potential association with the aging. Also, we delved into the underlying mechanisms. Our findings revealed that TXNIP significantly augmented pyroptosis in HT-22 cells, primarily by enhancing the activation of the NLRP3 inflammasome and promoting the release of proinflammatory cytokines. Remarkably, as TXNIP levels increased, we observed a corresponding rise in the number of p16-positive cells, which is indicative of aging. Furthermore, we conducted experiments to modulate the improvement of TXNIP on NLRP3 inflammasome-induced pyroptosis, that is, the PI3K activator 740 Y-P and the PKA activator DC2797 inhibited the effect, while the PI3K inhibitor LY294002 and the PKA inhibitor H89 enhanced the effect. In conclusion, our study demonstrated that TXNIP regulates NLRP3 inflammasome-induced pyroptosis in HT-22 cells related to aging via the PI3K/Akt and cAMP/PKA pathways., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cyclic adenosine monophosphate (cAMP) is an intracellular second messenger that is derived from the conversion of adenosine triphosphate catalysed by adenylyl cyclase (AC). Protein kinase A (PKA), the main effector of cAMP, is a dimeric protein kinase consisting of two catalytic subunits and two regulatory subunits. When cAMP binds to the regulatory subunits of PKA, it leads to the dissociation and activation of PKA, which allows the catalytic subunit of PKA to phosphorylate target proteins, thereby regulating various physiological functions and metabolic processes in cellular function. Recent researches also implicate the involvement of cAMP-PKA signaling in the pathologenesis of anxiety disorder. However, there are still debates on the prevention and treatment of anxiety disorders from this signaling pathway. To review the function of cAMP-PKA signaling in anxiety disorder, we searched the publications with the keywords including "cAMP", "PKA" and "Anxiety" from Pubmed, Embase, Web of Science and CNKI databases. The results showed that the number of publications on cAMP-PKA pathway in anxiety disorder tended to increase. Bioinformatics results displayed a close association between the cAMP-PKA pathway and the occurrence of anxiety. Mechanistically, cAMP-PKA signaling could influence brain-derived neurotrophic factor and neuropeptide Y and participate in the regulation of anxiety. cAMP-PKA signaling could also oppose the dysfunctions of gamma-aminobutyric acid (GABA), intestinal flora, hypothalamic-pituitary-adrenal axis, neuroinflammation, and signaling proteins (MAPK and AMPK) in anxiety. In addition, chemical agents with the ability to activate cAMP-PKA signaling demonstrated therapy potential against anxiety disorders. This review emphasizes the central roles of cAMP-PKA signaling in anxiety and the targets of the cAMP-PKA pathway would be potential candidates for treatment of anxiety. Nevertheless, more laboratory investigations to improve the therapeutic effect and reduce the adverse effect, and continuous clinical research will warrant the drug development., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Asthma is a chronic airway inflammation that is considered a serious public health concern worldwide. Nobiletin (5,6,7,8,3',4'-hexamethyl flavonoid), an important compound isolated from several traditional Chinese medicines, especially Citri Reticulatae Pericarpium, is widely used for a number of indications, including cancer, allergic diseases, and chronic inflammation. However, the mechanism by which nobiletin exerts its anti-asthmatic effect remains unclear. In this research, we comprehensively demonstrated the anti-asthmatic effects of nobiletin in an animal model of asthma. It was found that nobiletin significantly reduced the levels of inflammatory cells and cytokines in mice and alleviated airway hyperresponsiveness. To explore the target of nobiletin, we identified PDE4B as the target of nobiletin through pharmacophore modeling, molecular docking, molecular dynamics simulation, SPR, and enzyme activity assays. Subsequently, it was found that nobiletin could activate the cAMP-PKA-CREB signaling pathway downstream of PDE4B in mouse lung tissues. Additionally, we studied the anti-inflammatory and anti-airway remodeling effects of nobiletin in LPS-induced RAW264.7 cells and TGF-β1-induced ASM cells, confirming the activation of the cAMP-PKA-CREB signaling pathway by nobiletin. Further validation in PDE4B-deficient RAW264.7 cells confirmed that the increase in cAMP levels induced by nobiletin depended on the inhibition of PDE4B. In conclusion, nobiletin exerts anti-asthmatic activity by targeting PDE4B and activating the cAMP-PKA-CREB signaling pathway.

The cAMP cascade is increasingly recognized to transduce physiological effects locally through spatially limited cAMP gradients. However, little is known about how adenylyl cyclase enzymes that initiate cAMP gradients are localized. Here we address this question in physiologically relevant striatal neurons and investigate how AC localization impacts downstream signaling function. We show that the major striatal AC isoforms are differentially sorted between ciliary and extraciliary domains of the plasma membrane, and that one isoform, AC9, is uniquely concentrated in endosomes. We identify key sorting determinants in the N-terminal cytoplasmic domain responsible for isoform-specific localization. We further show that AC9-containing endosomes accumulate activated dopamine receptors and form an elaborately intertwined network with juxtanuclear PKA stores bound to Golgi membranes. Finally, we provide evidence that endosomal localization enables AC9 to selectively elevate PKA activity in the nucleus relative to the cytoplasm. Together, these results reveal a precise spatial landscape of the cAMP cascade in neurons and a key role of AC localization in directing downstream PKA signaling to the nucleus., (© 2024. The Author(s).)

Background: The KCNQ1+KCNE1 (I Ks ) potassium channel plays a crucial role in cardiac adaptation to stress, in which β-adrenergic stimulation phosphorylates the I Ks channel through the cyclic adenosine monophosphate (cAMP)/PKA (protein kinase A) pathway. Phosphorylation increases the channel current and accelerates repolarization to adapt to an increased heart rate. Variants in KCNQ1 can cause long-QT syndrome type 1 (LQT1), and those with defective cAMP effects predispose patients to the highest risk of cardiac arrest and sudden death. However, the molecular connection between I Ks channel phosphorylation and channel function, as well as why high-risk LQT1 mutations lose cAMP sensitivity, remain unclear., Methods: Regular patch clamp and voltage clamp fluorometry techniques were utilized to record pore opening and voltage sensor movement of wild-type and mutant KCNQ1/I Ks channels. The clinical phenotypic penetrance of each LQT1 mutation was analyzed as a metric for assessing their clinical risk. The patient-specific-induced pluripotent stem-cell model was used to test mechanistic findings in physiological conditions., Results: By systematically elucidating mechanisms of a series of LQT1 variants that lack cAMP sensitivity, we identified molecular determinants of I Ks channel regulation by phosphorylation. These key residues are distributed across the N-terminus of KCNQ1 extending to the central pore region of I Ks . We refer to this pattern as the I Ks channel PKA phosphorylation axis. Next, by examining LQT1 variants from clinical databases containing 10 579 LQT1 carriers, we found that the distribution of the most high-penetrance LQT1 variants extends across the I Ks channel PKA phosphorylation axis, demonstrating its clinical relevance. Furthermore, we found that a small molecule, ML277, which binds at the center of the phosphorylation axis, rescues the defective cAMP effects of multiple high-risk LQT1 variants. This finding was then tested in high-risk patient-specific induced pluripotent stem cell-derived cardiomyocytes, where ML277 remarkably alleviates the beating abnormalities., Conclusions: Our findings not only elucidate the molecular mechanism of PKA-dependent I Ks channel phosphorylation but also provide an effective antiarrhythmic strategy for patients with high-risk LQT1 variants., Competing Interests: None.

Hypertrophic cardiomyopathy (HCM) arises from a pathogenic variant in the gene responsible for encoding the myocardium-associated protein. Forskolin (FSK), a labdane diterpene isolated from Sphingomonas capillaris, exhibits diverse pharmacological effects, including bronchospasm relief, intraocular pressure reduction, and glaucoma treatment. However, whether FSK could regulate HCM and its associated mechanism remains unclear. Here, we discovered that FSK could mitigate cardiac hypertrophy in two HCM mouse models (Myh6 R404Q and Tnnt2 R109Q ) in vivo. Additionally, FSK could prevent norepinephrine (NE)-induced cardiomyocyte hypertrophy in vitro. It reversed cardiac dysfunction, reduced enlarged cell size, and downregulated the expression of hypertrophy-related genes. We further demonstrated that FSK's mechanism in alleviating HCM relied on the activation of ADCY6. In conclusion, our findings demonstrate that FSK alleviates hypertrophic cardiomyopathy by modulating the ADCY6/cAMP/PKA pathway, suggesting that FSK holds promise as a therapeutic agent for HCM., Competing Interests: Declaration of competing interest No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Adrenergic modulation of voltage gated Ca 2+ currents is a context specific process. In the heart Ca v 1.2 channels initiate excitation-contraction coupling. This requires PKA phosphorylation of the small GTPase Rad (Ras associated with diabetes) and involves direct phosphorylation of the Ca v 1.2 α 1 subunit at Ser1700. A contributing factor is the proximity of PKA to the channel through association with A-kinase anchoring proteins (AKAPs). Disruption of PKA anchoring by the disruptor peptide AKAP-IS prevents upregulation of Ca v 1.2 currents in tsA-201 cells. Biochemical analyses demonstrate that Rad does not function as an AKAP. Electrophysiological recording shows that channel mutants lacking phosphorylation sites (Ca v 1.2 STAA) lose responsivity to the second messenger cAMP. Measurements in cardiomyocytes isolated from Rad -/- mice show that adrenergic activation of Ca v 1.2 is attenuated but not completely abolished. Whole animal electrocardiography studies reveal that cardiac selective Rad KO mice exhibited higher baseline left ventricular ejection fraction, greater fractional shortening, and increased heart rate as compared to control animals. Yet, each parameter of cardiac function was slightly elevated when Rad -/- mice were treated with the adrenergic agonist isoproterenol. Thus, phosphorylation of Ca v 1.2 and dissociation of phospho-Rad from the channel are local cAMP responsive events that act in concert to enhance L-type calcium currents. This convergence of local PKA regulatory events at the cardiac L-type calcium channel may permit maximal β-adrenergic influence on the fight-or-flight response., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Torreya grandis (T. grandis) oil has been reported to alleviate symptoms of slow transit constipation (STC). However, the impact of sciadonic acid (SA), a distinctive fatty acid found in T. grandis oil, on the pathological progression of STC remains unclear. This study aimed to evaluate the effect of SA on STC and uncover the underlying mechanisms. The STC model was established by feeding Balb/c mice with loperamide. After 2 weeks of intervention, SA significantly improved weight loss and intestinal motility decline induced by STC, along with enhancing plasma indices and reducing colon pathological damage. SA effectively reversed the STC-induced decrease in the 5-HT4/cAMP/PKA/AQP4 signaling pathway genes and expression. Furthermore, 16S rRNA analysis demonstrated that SA mitigated the imbalance of the intestinal microbiota induced by STC, by reducing the ratio of Firmicutes to Bacteroidetes (F/B) and increasing the abundance of beneficial bacteria such as Akkermansia. In conclusion, SA intervention alleviated colonic dysfunction in STC mice. The activation of the SA-mediated 5-HT4/cAMP/PKA/AQP4 signaling pathway may serve as a potential target for STC treatment. These findings suggest that SA holds promise as a treatment option for STC and could potentially be extended to other related gut diseases for further investigation., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Premature ovarian insufficiency (POI) presents a substantial challenge to women's physiological and psychological well-being. Hormone replacement therapy, as the preferred therapeutic approach, involves solely exogenous supplementation of estrogen. Moxibustion, a traditional Chinese external treatment, has been investigated in our previous studies. It not only improves hormone levels and clinical symptoms in POI patients but also safeguards ovarian reserve. This study aims to explore the regulatory mechanisms by which moxibustion modulates hormone levels and restores ovarian function in POI. A POI rat model was established using cyclophosphamide, and moxibustion treatment was applied at acupoints "CV4" and "SP6" for a total of four courses. Subsequently, ovaries from each group were subjected to transcriptome sequencing (Bulk RNA-seq). Target pathways and key genes were selected through enrichment analysis and GSVA scoring, with validation using various techniques including electron microscopy, ELISA, Western blot, and immunohistochemistry. The results demonstrated that moxibustion restored the estrous cycle in POI rats, improved sex hormone levels, reduced the number of atretic follicles, and increased the count of dominant follicles (P<0.05). Bulk RNA-seq analysis revealed that moxibustion downregulated pathways associated with ovarian dysfunction, infertility, and immune responses, upregulated pathways related to follicular development and ovarian steroidogenesis. Furthermore, our data confirmed that moxibustion significantly increased the number of ovarian granulosa cells (GCs) and upregulated the expression of proteins related to steroidogenesis in GCs, including FSHR, P450 arom, cAMP, PKA, and CREB (P<0.05), with no significant effect observed on proteins related to steroidogenesis in theca cells. These outcomes aligned with the RNA-seq results. In conclusion, these findings propose that moxibustion enhances steroidogenesis in GCs through the activation of the cAMP/PKA/CREB pathway, consequently improving impaired ovarian function in POI rats. This study provides robust evidence supporting moxibustion as a targeted intervention for treating POI by specifically regulating steroidogenesis in GCs., Competing Interests: Declaration of Competing Interest All authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Taste receptor type 1 subunit 3 (T1R3) is a G protein-coupled receptor encoded by the TAS1R3 gene that can be specifically activated by certain sweeteners or umami agents for sweet/umami recognition. T1R3 is a potential target for regulating male reproduction. However, studies on the impact of non-nutritive sweeteners on reproduction are limited. In the present study, we evaluated the impact of the non-nutritive sweeteners (saccharin sodium, sucralose and acesulfame-K) on testosterone synthesis in testicular Leydig cells of Xiang pigs by comparing the relative abundance of mRNA transcripts and protein expression of T1R3, steroidogenic related factors, and intracellular cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), as well as testosterone levels using Western blotting, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). To clarify the specific mechanism, a dual luciferase assay was used to uncover the relationship between the transcription factors and steroidogenic enzyme. The acute intratesticular injection of a typical non-nutritive sweeteners was conducted to verify this impact in mouse. The results showed that saccharin sodium not only enhanced T1R3 expression in Leydig cells of Xiang pigs, but also caused significant increases in testosterone, cAMP, PKA, phosphorylation of specificity protein 1 (p-SP1), total protein of specificity protein 1 (SP1), steroidogenic acute regulatory protein (StAR), and 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD1) (P < 0.05). Similarly, treatment of Leydig cells with sucralose and acesulfame-K also increased testosterone level, protein expression of T1R3, 17-α-hydroxylase/17, 20-lyase (CYP17A1), and 3β-HSD1 (P < 0.05). Treatment with SQ22536 (an adenylate cyclas inhibitor) or H89 (a PKA inhibitor) significantly reduced saccharin sodium-induced protein levels of p-SP1, StAR, CYP17A1, and 3β-HSD1 (P < 0.05). In addition, a dual luciferase assay further demonstrated that SP1 significantly increased the promoter activity of CYP17A1 (P < 0.05). When mouse testes were injected with saccharin sodium, T1R3, p-SP1, CYP17A1, and 3β-HSD1 were upregulated, leading to a significant testicular increase in testosterone and cAMP levels (P < 0.05). These results suggest a mechanism by which the taste receptor T1R3 regulates testosterone production, and this mechanism may be linked to the cAMP-PKA pathway. Understanding the interrelationship between T1R3 and the cAMP-PKA-SP1 pathway contributes to clarify the regulatory mechanisms of male reproduction., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Leydig cells are the main testosterone-producing cells in males. During androgen synthesis, cholesterol enters the mitochondria via the STAR protein and is converted into pregnenolone by the CYP11A1 enzyme. This steroid is then exported from the mitochondria to be metabolized to progesterone by the HSD3B1 enzyme in the endoplasmic reticulum. In this study, we used 3'Tag-RNA-Seq to identify progesterone-regulated genes in MA-10 Leydig cells. Our results indicate that high concentrations of progesterone (30 μM) are involved in a negative feedback loop that inhibits cAMP/PKA-dependent activation of Star and Cyp11a1 expression and participate in cAMP/PKA-dependent down-regulation of genes related to the metabolism of steroid hormones. Linked to activation of the MAPK signaling pathway, endoplasmic reticulum stress and apoptosis, most of the genes encoding bZIP transcription factors are upregulated by progesterone in MA-10 Leydig cells. However, only DDIT3 protein levels are increased in response to progesterone in MA-10 Leydig cells. Like normal Leydig cells, MA-10 cells very weakly express the classical nuclear receptor for progesterone, suggesting that gene regulation by progesterone is rather mediated by one of the non-classical membrane receptors for progesterone However, current findings suggest that the inhibitory effect of progesterone on STAR protein increase in response to forskolin is not dependent on PGRMC1/2 or PAQR9. Furthermore, the increase in progesterone synthesis in response to activation of the cAMP/PKA pathway is rather inhibited by siRNA-mediated knockdown of PAQR9. Overall, this study shows that progesterone produced by Leydig cells participates in the regulation of steroidogenesis through autocrine action involving negative feedback upon activation of the cAMP/PKA pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Reduction of inflammatory damage and inhibition of nucleus pulposus (NP) apoptosis are considered to be the main effective therapy idea to reverse the intervertebral disc degeneration (IDD) and alleviate the chronic low back pain. The adenosine A2A receptor (A2AR), as a member of G protein-coupled receptor families, plays an important role in the anti-inflammation and relieving pain. So far, the impact of A2AR on IDD therapy is unclear. The aim of this study was to explore the role of Adenosine A 2A receptor (A 2A R) in the intervertebral disc degeneration (IDD) and clarify potential mechanism., Materials and Methods: IL-1β and acupuncture was used to establish IDD model rats. A 2A R agonist CGS-21680 and A 2A R antagonist SCH442416 were used to investigate the therapeutical effects for IDD. Histological examination, western blotting analysis and RT-PCR were employed to evaluate the the association between A 2A R and cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway., Results: A 2A R activity of the intervertebral disc tissues was up-regulated in feedback way, and cAMP, PKA and CREB expression were also increased. But in general, IL-1β-induced IDD promoted the significant up-regulation the expression of inflammatory factors. The nucleus pulposus (NP) inflammation was exacerbated in result of MMP3 and Col-II decline through activating NF-κB signaling pathway. A 2A R agonist CGS-21680 exhibited a disc protective effect through significantly increasing A 2A R activity, then further activated cAMP/PKA signaling pathway with attenuating the release of TNF-α and IL-6 via down-regulating NF-κB. In contrast, SCH442416 inhibited A 2A R activation, consistent with lower expression levels of cAMP and PKA, further leading to the acceleration of IDD., Conclusions: The activation of A 2A R can prevent inflammatory responses and mitigates degradation of IDD thus suggest a potential novel therapeutic strategy of IDD., (© 2024. The Author(s).)

Glutamate excitotoxicity is involved in retinal ganglion cell (RGC) death in various retinal degenerative diseases, including ischemia-reperfusion injury and glaucoma. Excitotoxic RGC death is caused by both direct damage to RGCs and indirect damage through neuroinflammation of retinal glial cells. Omidenepag (OMD), a novel E prostanoid receptor 2 (EP2) agonist, is a recently approved intraocular pressure-lowering drug. The second messenger of EP2 is cyclic adenosine monophosphate (cAMP), which activates protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). In this study, we investigated the neuroprotective effects of OMD on excitotoxic RGC death by focusing on differences in cAMP downstream signaling from the perspective of glia-neuron interactions. We established a glutamate excitotoxicity model in vitro and NMDA intravitreal injection model in vivo. In vitro, rat primary RGCs were used in an RGC survival rate assay. MG5 cells (mouse microglial cell line) and A1 cells (astrocyte cell line) were used for immunocytochemistry and Western blotting to evaluate the expressions of COX-1/2, PKA, Epac1/2, pCREB, cleaved caspase-3, inflammatory cytokines, and neurotrophic factors. Mouse retinal specimens underwent hematoxylin and eosin staining, flat-mounted retina examination, and immunohistochemistry. OMD significantly suppressed excitotoxic RGC death, cleaved caspase-3 expression, and activated glia both in vitro and in vivo. Moreover, it inhibited Epac1 and inflammatory cytokine expression and promoted COX-2, pCREB, and neurotrophic factor expression. OMD may have neuroprotective effects through inhibition of the Epac pathway and promotion of the COX-2-EP2-cAMP-PKA pathway by modulating glia-neuron interaction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background and Purpose: In human airway smooth muscle (hASM) cells, not all receptors stimulating cAMP production elicit the same effects. This can only be explained if cAMP movement throughout the cell is restricted, yet the mechanisms involved are not fully understood. Phosphodiesterases (PDEs) contribute to compartmentation of many cAMP responses, but PDE activity alone is predicted to be insufficient if cAMP is otherwise freely diffusible. We tested the hypothesis that buffering of cAMP by protein kinase A (PKA) associated with A kinase anchoring proteins (AKAPs) slows cAMP diffusion and that this contributes to receptor-mediated, compartmentalized responses., Experimental Approach: Raster image correlation spectroscopy (RICS) was used to measure intracellular cAMP diffusion coefficients and evaluate the contribution of PKA-AKAP interactions. Western blotting and immunocytochemistry were used to identify the AKAPs involved. RNA interference was used to down-regulate AKAP expression and determine its effects on cAMP diffusion. Compartmentalized cAMP responses were measured using fluorescence resonance energy transfer (FRET) based biosensors., Key Results: Cyclic AMP movement was significantly slower than that of free-diffusion in hASM cells, and disrupting PKA-AKAP interactions significantly increased the diffusion coefficient. PKA associated with the outer mitochondrial membrane appears to play a prominent role in this effect. Consistent with this idea, knocking down expression of D-AKAP2, the primary mitochondrial AKAP, increased cAMP diffusion and disrupted compartmentation of receptor-mediated responses., Conclusion and Implications: Our results confirm that AKAP-anchored PKA contributes to the buffering of cAMP and is consequential in the compartmentation of cAMP responses in hASM cells., (© 2024 British Pharmacological Society.)

Acute cholestatic liver injury (ACLI) is a disease associated with bile duct obstruction that causes liver inflammation and apoptosis. Although G protein-coupled bile acid receptor1 (Gpbar-1) has diverse metabolic roles, its involvement in ACLI-associated immune activation remains unclear. Liver tissues and blood samples from 20 patients with ACLI and 20 healthy individuals were analyzed using biochemical tests, H&E staining, western blotting, and immunohistochemistry to verify liver damage and expression of Gpbar-1. The expression of Gpbar-1, cAMP/PKA signaling, and the NLRP3 inflammasome was tested in wild-type (WT) and Gpbar-1 knockdown (si-Gpbar-1) mice with ACLI induced by bile duct ligation (BDL) and in primary Kupffer cells (KCs) with or without Gpbar-1-siRNA. The results showed that total bile acids and Gpbar-1 expressions were elevated in patients with ACLI. Gpbar-1 knockdown significantly worsened BDL-induced acute hepatic damage, inflammation, and liver apoptosis in vivo. Knockdown of Gpbar-1 heightened KC sensitivity to lipopolysaccharide (LPS) stimulation. Gpbar-1 activation inhibited LPS-induced pro-inflammatory responses in normal KCs but not in Gpbar-1-knockdown KCs. Notably, NLRP3-ASC inflammasome expression was effectively enhanced by Gpbar-1 deficiency. Additionally, Gpbar-1 directly increased intracellular cAMP levels and PKA phosphorylation, thus disrupting the NLRP3-ASC inflammasome. The pro-inflammatory characteristic of Gpbar-1 deficiency was almost neutralized by the NLRP3 inhibitor CY-09. In vitro, M1 polarization was accelerated in LPS-stimulated Gpbar-1-knockdown KCs. Therapeutically, Gpbar-1 deficiency exacerbated BDL-induced ACLI, which could be rescued by inhibition of the NLRP3-ASC inflammasome. Our study reveal that Gpbar-1 may act as a novel immune-mediated regulator of ACLI by inhibiting the NLRP3-ASC inflammasome., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests. All authors agree to the author list., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Aim: Apigenin, a natural bioflavonoid, is reported as an anti-diabetic agent since it possesses the ability to inhibit α-glucosidase activity, cause stimulation of insulin action and secretion, manage ROS, and prevent diabetes complications. Apigenin was identified as a new insulin secretagogue that enhances glucose-stimulated insulin secretion and seems like a better antidiabetic drug candidate. Here we explored the insulinotropic mechanism(s) of apigenin in vitro in mice islets and in vivo in diabetic rats., Methods: Size-matched pancreatic islets were divided into groups and incubated in the presence or absence of apigenin and agonists or antagonists of major insulin signaling pathways. The secreted insulin was measured by ELISA. The intracellular cAMP was estimated by cAMP acetylation assay. The acute and chronic effects of apigenin were evaluated in diabetic rats., Results: apigenin dose-dependently enhanced insulin secretion in isolated mice islets, and its insulinotropic effect was exerted at high glucose concentrations distinctly different from glibenclamide. Furthermore, apigenin amplified glucose-induced insulin secretion in depolarized and glibenclamide-treated islets. Apigenin showed no effect on intracellular cAMP concentration; however, an additive effect was observed by apigenin in both forskolin and IBMX-induced insulin secretion. Interestingly, H89, a PKA inhibitor, and U0126, a MEK kinase inhibitor, significantly inhibited apigenin-induced insulin secretion; however, no significant effect was observed by using ESI-05, an epac2 inhibitor. Apigenin improved glucose tolerance and increased glucose-stimulated plasma insulin levels in diabetic rats. Apigenin also lowered blood glucose in diabetic rats upon chronic treatment., Conclusion: Apigenin exerts glucose-stimulated insulin secretion by modulating the PKA-MEK kinase signaling cascade independent of K-ATP channels., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Melanoma, a cancer arising from melanocytes, requires a novel treatment strategy because of the ineffectiveness of conventional therapies in certain patients. Fustin is a flavanonol found in young fustic (Cotinus coggygria). However, little is known about its antimelanoma effects. Our study demonstrates that fustin suppresses the growth of B16 melanoma cells. Phalloidin staining of cytoskeletal actin revealed that fustin induced a conformational change in the actin structure of melanoma cells, accompanied by suppressed phosphorylation of myosin regulatory light chain 2 (MLC2), a regulator of actin structure. Furthermore, the protein kinase A (cAMP-dependent protein kinase) inhibitor H89 completely attenuated fustin-induced downregulation of phosphorylated myosin phosphatase targeting subunit 1, which is involved in dephosphorylation of MLC2. In a mouse model, administration of fustin suppressed tumor growth in B16 melanoma cells without adverse effects. In conclusion, our findings suggest that fustin effectively suppresses melanoma cell growth both in vitro and in vivo., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Estrogen excess in females has been linked to a diverse array of chronic and acute diseases. Emerging research shows that exposure to estrogen-like compounds such as bisphenol S leads to increases in 17β-estradiol levels, but the mechanism of action is unclear. The aim of this study was to reveal the underlying signaling pathway-mediated mechanisms, target site and target molecule of action of bisphenol S causing excessive estrogen synthesis. Human ovarian granulosa cells SVOG were exposed to bisphenol S at environmentally relevant concentrations (1 μg/L, 10 μg/L, and 100 μg/L) for 48 h. The results confirms that bisphenol S accumulates mainly on the cell membrane, binds to follicle stimulating hormone receptor (FSHR) located on the cell membrane, and subsequently activates the downstream cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signaling pathway, leading to enhanced conversion of testosterone to 17β-estradiol. This study deepens our knowledge of the mechanisms of environmental factors in pathogenesis of hyperestrogenism., (© 2024. The Author(s).)

This study aimed to elucidate the opioid mechanisms underlying dexamethasone-induced pain antihypersensitive effects in neuropathic rats. Dexamethasone (subcutaneous and intrathecal) and membrane-impermeable Dex-BSA (intrathecal) administration dose-dependently inhibited mechanical allodynia and thermal hyperalgesia in neuropathic rats. Dexamethasone and Dex-BSA treatments increased expression of dynorphin A in the spinal cords and primary cultured microglia. Dexamethasone specifically enhanced dynorphin A expression in microglia but not astrocytes or neurons. Intrathecal injection of the microglial metabolic inhibitor minocycline blocked dexamethasone-stimulated spinal dynorphin A expression; intrathecal minocycline, the glucocorticoid receptor antagonist Dex-21-mesylate, dynorphin A antiserum, and κ-opioid receptor antagonist GNTI completely blocked dexamethasone-induced mechanical antiallodynia and thermal antihyperalgesia. Additionally, dexamethasone elevated spinal intracellular cAMP levels, leading to enhanced phosphorylation of PKA, p38 MAPK and CREB. The specific adenylate cyclase inhibitor DDA, PKA inhibitor H89, p38 MAPK inhibitor SB203580 and CREB inhibitor KG-501 completely blocked dexamethasone-induced anti-neuropathic pain and increased microglial dynorphin A exprression. In conclusion, this study reveal that dexamethasone mitigateds neuropathic pain through upregulation of dynorphin A in spinal microglia, likely involving the membrane glucocorticoid receptor/cAMP/PKA/p38 MAPK/CREB signaling pathway., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

1,2-Dichloroethane (1,2-DCE) is a powerfully toxic neurotoxin, which is a common environmental pollutant. Studies have indicated that 1,2-DCE long-term exposure can result in adverse effects. Nevertheless, the precise mechanism remains unknown. In this study, behavioral results revealed that 1,2-DCE long-term exposure could cause anxiety and learning and memory ability impairment in mice. The contents of γ-aminobutyric acid (GABA) and glutamine (Gln) in mice's prefrontal cortex decreased, whereas that of glutamate (Glu) increased. With the increase in dose, the activities of glutamate decarboxylase (GAD) decreased and those of GABA transaminase (GABA-T) increased. The protein and mRNA expressions of GABA transporter-3 (GAT-3), vesicular GABA transporter (VGAT), GABA A receptor α2 (GABA A Rα2), GABA A Rγ2, K-Cl cotransporter isoform 2 (KCC2), GABA B receptor 1 (GABA B R1), GABA B R2, protein kinase A (PKA), cAMP-response element binding protein (CREB), p-CREB, brain-derived neurotrophic factor (BDNF), c-fos, c-Jun and the protein of glutamate dehydrogenase (GDH) and PKA-C were decreased, while the expression levels of GABA transporter-1 (GAT-1) and Na-K-2Cl cotransporter isoform 1 (NKCC1) were increased. However, there was no significant change in the protein content of succinic semialdehyde dehydrogenase (SSADH). The expressions of adenylate cyclase (AC) and cyclic adenosine monophosphate (cAMP) contents were also reduced. In conclusion, the results of this study show that exposure to 1,2-DCE could lead to anxiety and cognitive impairment in mice, which may be related to the disturbance of GABA metabolism and its receptors along with the cAMP-PKA-CREB pathway., Competing Interests: Declaration of Competing Interest Authors listed in this paper participated in the design, execution, and analysis of this paper. All other authors have read the manuscript and have agreed to submit it in its current form for consideration for publication in the Journal. The authors declare that there are no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Candida albicans is an opportunistic fungal pathogen that can switch between yeast and hyphal morphologies depending on the environmental cues it receives. The switch to hyphal form is crucial for the establishment of invasive infections. The hyphal form is also characterized by the cell surface expression of hyphae-specific proteins, many of which are GPI-anchored and important determinants of its virulence. The coordination between hyphal morphogenesis and the expression of GPI-anchored proteins is made possible by an interesting cross-talk between GPI biosynthesis and the cAMP-PKA signaling cascade in the fungus; a parallel interaction is not found in its human host. On the other hand, in the nonpathogenic yeast, Saccharomyces cerevisiae, GPI biosynthesis is shut down when filamentation is activated and vice versa. This too is achieved by a cross-talk between GPI biosynthesis and cAMP-PKA signaling. How are diametrically opposite effects obtained from the cross-talk between two reasonably well-conserved pathways present ubiquitously across eukarya? This Review attempts to provide a model to explain these differences. In order to do so, it first provides an overview of the two pathways for the interested reader, highlighting the similarities and differences that are observed in C. albicans versus the well-studied S. cerevisiae model, before going on to explain how the different mechanisms of regulation are effected. While commonalities enable the development of generalized theories, it is hoped that a more nuanced approach, that takes into consideration species-specific differences, will enable organism-specific understanding of these processes and contribute to the development of targeted therapies., Competing Interests: Conflict of interest The author declares that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Platelet activation is critical for haemostasis, but if unregulated can lead to pathological thrombosis. Endogenous platelet inhibitory mechanisms are mediated by prostacyclin (PGI 2 )-stimulated cAMP signalling, which is regulated by phosphodiesterase 3A (PDE3A). However, spatiotemporal regulation of PDE3A activity in platelets is unknown. Here, we report that platelets possess multiple PDE3A isoforms with seemingly identical molecular weights (100 kDa). One isoform contained a unique N-terminal sequence that corresponded to PDE3A1 in nucleated cells but with negligible contribution to overall PDE3A activity. The predominant cytosolic PDE3A isoform did not possess the unique N-terminal sequence and accounted for >99% of basal PDE3A activity. PGI 2 treatment induced a dose and time-dependent increase in PDE3A phosphorylation which was PKA-dependent and associated with an increase in phosphodiesterase enzymatic activity. The effects of PGI 2 on PDE3A were modulated by A-kinase anchoring protein (AKAP) disruptor peptides, suggesting an AKAP-mediated PDE3A signalosome. We identified AKAP7, AKAP9, AKAP12, AKAP13, and moesin expressed in platelets but focussed on AKAP7 as a potential PDE3A binding partner. Using a combination of immunoprecipitation, proximity ligation techniques, and activity assays, we identified a novel PDE3A/PKA RII/AKAP7 signalosome in platelets that integrates propagation and termination of cAMP signalling through coupling of PKA and PDE3A.

Cytoplasmic mislocalization and aggregation of TDP-43 protein are hallmarks of amyotrophic lateral sclerosis (ALS) and are observed in the vast majority of both familial and sporadic cases. How these two interconnected processes are regulated on a molecular level, however, remains enigmatic. Genome-wide screens for modifiers of the ALS-associated genes TDP-43 and FUS have identified the phospholipase D (Pld) pathway as a key regulator of ALS-related phenotypes in the fruit fly Drosophila melanogaster [M. W. Kankel et al. , Genetics 215 , 747-766 (2020)]. Here, we report the results of our search for downstream targets of the enzymatic product of Pld, phosphatidic acid. We identify two conserved negative regulators of the cAMP/PKA signaling pathway, the phosphodiesterase dunce and the inhibitory subunit PKA-R2 , as modifiers of pathogenic phenotypes resulting from overexpression of the Drosophila TDP-43 ortholog TBPH . We show that knockdown of either of these genes results in a mitigation of both TBPH aggregation and mislocalization in larval motor neuron cell bodies, as well as an amelioration of adult-onset motor defects and shortened lifespan induced by TBPH. We determine that PKA kinase activity is downstream of both TBPH and Pld and that overexpression of the PKA target CrebA can rescue TBPH mislocalization. These findings suggest a model whereby increasing cAMP/PKA signaling can ameliorate the molecular and functional effects of pathological TDP-43., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: This study investigated the relaxation effect of PGE2 on the ureter and its role in promoting calculi expulsion following calculi development., Methods: By using immunofluorescence and Western blot, we were able to locate EP receptors in the ureter. In vitro experiments assessed the impact of PGE2, receptor antagonists, and agonists on ureteral relaxation rate. We constructed a model of ureteral calculi with flowable resin and collected ureteral tissue from postoperative side of the ureter after obstruction surgery. Western blot analysis was used to determine the protein expression levels of EP receptors and the PGE2 terminal synthase mPGES-1. Additionally, PGE2 was added to smooth muscle cells to observe downstream cAMP and PKA changes., Results: The expression of EP2 and EP4 proteins in ureteral smooth muscle was verified by Western blot analysis. According to immunofluorescence, EP2 was primarily found on the cell membrane, while EP4 was found in the nucleus. In vitro, PGE2 induced concentration-dependent ureteral relaxation. Maximum diastolic rate was 70.94 ± 4.57% at a concentration of 30µM. EP2 antagonists hindered this effect, while EP4 antagonists did not. Obstructed ureters exhibited elevated mPGES-1 and EP2 protein expression (P < 0.01). Smooth muscle cells treated with PGE2 displayed increased cAMP and phosphorylated PKA., Conclusions: PGE2 binding to EP2 induces ureteral relaxation through the cAMP-PKA pathway. This will provide a new theoretical basis for the development of new therapeutic approaches for the use of PGE2 in the treatment of ureteral stones., (© 2024. The Author(s).)

Gonadotropin-releasing hormone (GnRH) is a key stimulator for gonadotropin secretion in the pituitary and its pivotal role in reproduction is well conserved in vertebrates. In fish models, GnRH can also induce prolactin (PRL) release, but little is known for the corresponding effect on PRL gene expression as well as the post-receptor signalling involved. Using grass carp as a model, the functional role of GnRH and its underlying signal transduction for PRL regulation were examined at the pituitary level. Using laser capture microdissection coupled with RT-PCR, GnRH receptor expression could be located in carp lactotrophs. In primary cell culture prepared from grass carp pituitaries, the native forms of GnRH, GnRH2 and GnRH3, as well as the GnRH agonist [D-Arg 6 , Pro 9 , NEt]-sGnRH were all effective in elevating PRL secretion, PRL mRNA level, PRL cell content and total production. In pituitary cells prepared from the rostral pars distalis, the region in the carp pituitary enriched with lactotrophs, GnRH not only increased cAMP synthesis with parallel CREB phosphorylation and nuclear translocation but also induced a rapid rise in cytosolic Ca 2+ by Ca 2+ influx via L-type voltage-sensitive Ca 2+ channel (VSCC) with subsequent CaM expression and NFAT 2 dephosphorylation. In carp pituitary cells prepared from whole pituitaries, GnRH-induced PRL secretion was reduced/negated by inhibiting cAMP/PKA, PLC/PKC and Ca 2+ /CaM/CaMK-II pathways but not the signalling events via IP 3 and CaN/NFAT. The corresponding effect on PRL mRNA expression, however, was blocked by inhibiting cAMP/PKA/CREB/CBP and Ca 2+ /CaM/CaN/NFAT 2 signalling but not PLC/IP 3 /PKC pathway. At the pituitary cell level, activation of cAMP/PKA pathway could also induce CaM expression and Ca 2+ influx via VSCC with parallel rises in PRL release and gene expression in a Ca 2+ /CaM-dependent manner. These findings, as a whole, suggest that the cAMP/PKA-, PLC/PKC- and Ca 2+ /CaM-dependent cascades are differentially involved in GnRH-induced PRL secretion and PRL transcript expression in carp lactotrophs. During the process, a functional crosstalk between the cAMP/PKA- and Ca 2+ /CaM-dependent pathways may occur with PRL release linked with CaMK-II and PKC activation and PRL gene transcription caused by nuclear action of CREB/CBP and CaN/NFAT 2 signalling., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Li, Ye, He, Ko, Cheng, Chan and Wong.)

Gut microbiota variances reflecting the severity type 2 diabetes mellitus (T2DM). Achyranthes bidentata polysaccharide (ABP) can regulate gut microbiota. However, the hypoglycemic effect and underlying mechanism of ABP remain unclear. Herein, we characterized the structure of ABP and revealed the hypoglycemic effect of ABP in mice with T2DM. ABP repaired the intestinal barrier in T2DM mice and regulated the composition and abundance of gut microbiota, especially increasing bacteria which producing short-chain fatty acids (SCFAs), then increasing glucagon-like peptide-1 (GLP-1) level. The abundance of these bacteria was positively correlated with blood lipid and INS levels, negatively correlated with FBG levels. Colon transcriptome data and immunohistochemistry demonstrated that the alleviating T2DM effect of ABP was related to activation of the GLP-1/GLP-1 receptor (GLP-1R)/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP-response element binding protein (CREB)/INS pathway. Fecal microbiota transplantation (FMT) confirmed the transmissible efficacy of ABP through gut microbiota. Overall, our research shows that ABP plays a hypoglycemic role by increasing gut microbiota-derived SCFAs levels, and activating the GLP-1/GLP-1R/cAMP/PKA/CREB/INS pathway, emphasizing ABP as promising T2DM therapeutic candidates., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: To explore the molecular mechanism of Astragaloside IV (AS-IV) in alleviating renal fibrosis by inhibiting Urotensin II-induced pyroptosis and epithelial-mesenchymal transition of renal tubular epithelial cells., Methods: Forty SD rats were randomly divided into control group without operation: gavage with 5ml/kg/d water for injection and UUO model group: gavage with 5ml/kg/d water for injection; UUO+ AS-IV group (gavage with AS-IV 20mg/kg/d; and UUO+ losartan potassium group (gavage with losartan potassium 10.3mg/kg/d, with 10 rats in each group. After 2 weeks, Kidney pathology, serum Urotensin II, and cAMP concentration were detected, and the expressions of NLRP3, GSDMD-N, Caspase-1, and IL-1β were detected by immunohistochemistry. Rat renal tubular epithelial cells were cultured in vitro, and different concentrations of Urotensin II were used to intervene for 24h and 48h. Cell proliferation activity was detected using the CCK8 assay. Suitable concentrations of Urotensin II and intervention time were selected, and Urotensin II receptor antagonist (SB-611812), inhibitor of PKA(H-89), and AS-IV (15ug/ml) were simultaneously administered. After 24 hours, cells and cell supernatants from each group were collected. The cAMP concentration was detected using the ELISA kit, and the expression of PKA, α-SMA, FN, IL-1β, NLRP3, GSDMD-N, and Caspase-1 was detected using cell immunofluorescence, Western blotting, and RT-PCR., Results: Renal tissue of UUO rats showed renal interstitial infiltration, tubule dilation and atrophy, renal interstitial collagen fiber hyperplasia, and serum Urotensin II and cAMP concentrations were significantly higher than those in the sham operation group (p <0.05). AS-IV and losartan potassium intervention could alleviate renal pathological changes, and decrease serum Urotensin II, cAMP concentration levels, and the expressions of NLRP3, GSDMD-N, Caspase-1, and IL-1β in renal tissues (p <0.05). Urotensin II at a concentration of 10-8 mol/L could lead to the decrease of cell proliferation, (p<0.05). Compared with the normal group, the cAMP level and the PKA expression were significantly increased (p<0.05). After intervention with AS-IV and Urotensin II receptor antagonist, the cAMP level and the expression of PKA were remarkably decreased (p<0.05). Compared with the normal group, the expression of IL-1β, NLRP3, GSDMD-N, and Caspase-1 in the Urotensin II group was increased (p<0.05), which decreased in the AS-IV and H-89 groups., Conclusion: AS-IV can alleviate renal fibrosis by inhibiting Urotensin II-induced pyroptosis of renal tubular epithelial cells by regulating the cAMP/PKA signaling pathway., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The cAMP-PKA signaling pathway plays a crucial role in sensing and responding to nutrient availability in the fission yeast Schizosaccharomyces pombe. This pathway monitors external glucose levels to control cell growth and sexual differentiation. However, the temporal dynamics of the cAMP-PKA pathway in response to external stimuli remains unclear mainly due to the lack of tools to quantitatively visualize the activity of the pathway. Here, we report the development of the kinase translocation reporter (KTR)-based biosensor spPKA-KTR1.0, which allows us to measure the dynamics of PKA activity in fission yeast cells. The spPKA-KTR1.0 is derived from the transcription factor Rst2, which translocates from the nucleus to the cytoplasm upon PKA activation. We found that spPKA-KTR1.0 translocates between the nucleus and cytoplasm in a cAMP-PKA pathway-dependent manner, indicating that the spPKA-KTR1.0 is a reliable indicator of the PKA activity in fission yeast cells. In addition, we implemented a system that simultaneously visualizes and manipulates the cAMP-PKA signaling dynamics by introducing bPAC, a photoactivatable adenylate cyclase, in combination with spPKA-KTR1.0. This system offers an opportunity for investigating the role of the signaling dynamics of the cAMP-PKA pathway in fission yeast cells with higher temporal resolution., (© 2024 The Authors. Yeast published by John Wiley & Sons Ltd.)

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal illness in humans and animals, induced by enterotoxins produced by these pathogens. Despite the crucial role of neutrophils in combatting bacterial infections, our understanding of how enterotoxins impact neutrophil function is limited. To address this knowledge gap, we used heat-labile enterotoxin (LT) and heat-stable enterotoxin a (STa) to investigate their impact on the effector functions of neutrophils. Our study reveals that pSTa does not exert any discernible effect on the function of neutrophils. In contrast, LT altered the migration and phagocytosis of neutrophils and induced the production of inflammatory factors via activation of cAMP/PKA and ERK1/2 signaling. LT also attenuated the release of neutrophil extracellular traps by neutrophils via the PKA signaling pathway. Our findings provide novel insights into the impact of LT on neutrophil function, shedding light on the underlying mechanisms that govern its immunoregulatory effects. This might help ETEC in subverting the immune system and establishing infection.

Background: It has been proven that vasoactive intestinal peptide (VIP) was involved in the pathogenesis of prostate cancer. Cardin et al . found that by an alanine scan, the heparin- binding site on VIP was exactly the same sequence in VIP and its receptor. Therefore, heparin could competitively block the binding of VIP and its receptor. However, the structure-activity relationship between heparin and VIP has not been reported, especially in terms of the sequence and sulfation patterns of heparin oligosaccharides upon binding to VIP., Objective: A variety of experiments were designed to study the binding process and structure-activity relationship between heparin oligosaccharides and VIP., Methods: Heparin was enzymatically digested and purified to produce heparin oligosaccharides, and the structures were characterized by NMR. The binding capacity between heparin oligosaccharides and VIP was analyzed by GMSA and ITC experiments. The binding between heparin oligosaccharides and VIP was simulated using a molecular docking program to show the complex. ELISA assay was used to investigate the effect of non-anticoagulant heparin oligosaccharides on the VIP-mediated cAMP/PKA signaling pathway in vitro ., Results: The results indicated that both the length and the sulfation pattern of heparin oligosaccharides affected its binding to VIP. VIP could induce the expression of cAMP at a higher level in PC3 cells, which could be regulated by the interaction of heparin oligosaccharides and VIP., Conclusion: The binding between heparin oligosaccharides and VIP could block the binding between VIP and its receptor on tumor cells. Downloading the regulation of the expression level of cAMP could possibly further affect the subsequent activation of PKA. These non-anticoagulant heparin oligosaccharides may block the VIP-mediated cAMP/PKA signaling pathway and thus exert their antitumor activity., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

We studied the involvement of cAMP and PKA in the regulation of the secretion of neurotrophic growth factors by macro-and microglial cells in the model of ethanol-induced neurodegeneration in vitro and in vivo. The stimulating role of cAMP in the secretion of neurotrophins by intact astrocytes and oligodendrocytes was shown, while PKA does not participate in this process. On the contrary, the inhibitory role of cAMP (implemented via PKA activation) in the production of neurogenesis stimulators by microglial cells under conditions of optimal vital activity was found. Under the influence of ethanol, the role of cAMP and PKA in the production of growth factors by macroglial cells was considerably changed. The involvement of PKA in the cAMP-dependent signaling pathways and inversion of the role of this signaling pathway in the implementation of the neurotrophic secretory function of astrocytes and oligodendrocytes, respectively, directly exposed to ethanol in vitro were noted. Long-term exposure of the nervous tissue to ethanol in vivo led to the loss of the stimulating role of cAMP/PKA signaling on neurotrophin secretion by macroglial cells without affecting its inhibitory role in the regulation of this function in microglial cells., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

cAMP, a key player in many physiological processes, was classically considered to originate solely from the plasma membrane (PM). This view was recently challenged by observations showing that upon internalization GsPCRs can sustain signaling from endosomes and/or the trans-Golgi network (TGN). In this new view, after the first PM-generated cAMP wave, the internalization of GsPCRs and ACs generates a second wave that was strictly associated with nuclear transcriptional events responsible for triggering specific biological responses. Here, we report that the endogenously expressed TSHR, a canonical GsPCR, triggers an internalization-dependent, calcium-mediated nuclear sAC activation that drives PKA activation and CREB phosphorylation. Both pharmacological and genetic sAC inhibition, which did not affect the cytosolic cAMP levels, blunted nuclear cAMP accumulation, PKA activation, and cell proliferation, while an increase in nuclear sAC expression significantly enhanced cell proliferation. Furthermore, using novel nuclear-targeted optogenetic actuators, we show that light-stimulated nuclear cAMP synthesis can mimic the proliferative action of TSH by activating PKA and CREB. Therefore, based on our results, we propose a novel three-wave model in which the "third" wave of cAMP is generated by nuclear sAC. Despite being downstream of events occurring at the PM (first wave) and endosomes/TGN (second wave), the nuclear sAC-generated cAMP (third wave) is sufficient and rate-limiting for thyroid cell proliferation.

The cyclic adenosine monophosphate (cAMP) signalling pathway plays an important role in the regulation of the development and pathogenicity of filamentous fungi. cAMP-dependent protein kinase A (PKA) is the conserved element downstream of cAMP, and its diverse mechanisms in multiple filamentous fungi are not well known yet. In the present study, gene knockout mutants of two catalytic subunits of PKA (PKA-C) in Setosphaeria turcica were created to illustrate the regulatory mechanisms of PKA-Cs on the development and pathogenicity of S. turcica. As a result, StPkaC2 was proved to be the main contributor of PKA activity in S. turcica. In addition, it was found that both StPkaC1 and StPkaC2 were necessary for conidiation and invasive growth, while only StPkaC2 played a negative role in the regulation of filamentous growth. We reveal that only StPkaC2 could interact with the transcription factor StEfg1, and it inhibited the transcription of StRAB1, a Rab GTPase homologue coding gene in S. turcica, whereas StPkaC1 could specifically interact with a transcriptional regulator StFlo8, which could rescue the transcriptional inhibition of StEfg1 on StRAB1. We also demonstrated that StRAB1 could positively influence the biosynthesis of chitin in hyphae, thus changing the filamentous growth. Our findings clarify that StPkaC2 participates in chitin biosynthesis to modulate mycelium development by targeting the Efg1-mediated transcription of StRAB1, while StFlo8, interacting with StPkaC1, acts as a negative regulator during this process., (© 2022 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Eukaryotic cells have developed a complex circuitry of signalling molecules which monitor changes in their intra- and extracellular environments. One of the most widely studied signalling pathways is the highly conserved cyclic AMP (cAMP)/protein kinase A (PKA) pathway, which is a major glucose sensing circuit in the yeast Saccharomyces cerevisiae. PKA activity regulates diverse targets in yeast, positively activating the processes that are associated with rapid cell growth (e.g., fermentative metabolism, ribosome biogenesis and cell division) and negatively regulating the processes that are associated with slow growth, such as respiratory growth, carbohydrate storage and entry into stationary phase. As in higher eukaryotes, yeast has evolved complexity at the level of the PKA catalytic subunit, and Saccharomyces cerevisiae expresses three isoforms, denoted Tpk1-3. Despite evidence for isoform differences in multiple biological processes, the molecular basis of PKA signalling specificity remains poorly defined, and many studies continue to assume redundancy with regards to PKA-mediated regulation. PKA has canonically been shown to play a key role in fine-tuning the cellular response to diverse stressors; however, recent studies have now begun to interrogate the requirement for individual PKA catalytic isoforms in coordinating distinct steps in stress response pathways. In this review, we discuss the known non-redundant functions of the Tpk catalytic subunits and the evolving picture of how these isoforms establish specificity in the response to different stress conditions.

Cancer is one of the most common causes of death globally. Despite extensive research and considerable advances in cancer therapy, the fundamentals of the disease remain unclear. Understanding the key signaling mechanisms that cause cancer cell malignancy may help to uncover new pharmaco-targets. Cyclic adenosine monophosphate (cAMP) regulates various biological functions, including those in malignant cells. Understanding intracellular second messenger pathways is crucial for identifying downstream proteins involved in cancer growth and development. cAMP regulates cell signaling and a variety of physiological and pathological activities. There may be an impact on gene transcription from protein kinase A (PKA) as well as its downstream effectors, such as cAMP response element-binding protein (CREB). The position of CREB downstream of numerous growth signaling pathways implies its oncogenic potential in tumor cells. Tumor growth is associated with increased CREB expression and activation. PKA can be used as both an onco-drug target and a biomarker to find, identify, and stage tumors. Exploring cAMP effectors and their downstream pathways in cancer has become easier using exchange protein directly activated by cAMP (EPAC) modulators. This signaling system may inhibit or accelerate tumor growth depending on the tumor and its environment. As cAMP and its effectors are critical for cancer development, targeting them may be a useful cancer treatment strategy. Moreover, by reviewing the material from a distinct viewpoint, this review aims to give a knowledge of the impact of the cAMP signaling pathway and the related effectors on cancer incidence and development. These innovative insights seek to encourage the development of novel treatment techniques and new approaches.

Depression is a common neuropsychiatric disorder characterized by persistent depressed mood and causes serious socioeconomic burden worldwide. Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis, deficiency of monoamine transmitters, neuroinflammation and abnormalities of the gut flora are strongly associated with the onset of depression. The cyclic AMP (cAMP)/protein kinase A (PKA) cascade, a major cross-species cellular signaling pathway, is supposed as important player and regulator of depression onset by controlling synaptic plasticity, cytokinesis, transcriptional regulation and HPA axis. In the central nervous system, the cAMP-PKA cascade can dynamically shape neural circuits by enhancing synaptic plasticity, and affect K + channels by phosphorylating Kir4.1, thereby regulating neuronal excitation. The reduction of cAMP-PKA cascade affects neuronal excitation as well as synaptic plasticity, ultimately leading to pathological outcome of depression, while activation of cAMP-PKA cascade would provide a rapid antidepressant effect. In this review, we proposed to reconsider the function of cAMP-PKA cascade, especially in the rapid antidepressant effect. Local activation or indirect activation of PKA through adjusting anchor proteins would provide new idea for acute treatment of depression., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Adult mammalian cardiomyocytes (CMs) lose their proliferative potential due to cell-cycle withdrawal and polyploidization and fail to mount a proliferative response to regenerate new CMs after cardiac injury. The decline in the proliferative potential of mammalian CMs occurs in the neonatal period when the endocrine system undergoes drastic changes for adaptation to extra-uterine life. There is an increase in circulating glucocorticoid (GC) levels shortly after birth in mammals, and thus, we sought to determine the roles and mechanisms of GCs in regulating CM proliferation. Here, we showed that GCs suppressed CM proliferation in vitro and in vivo, decreased the total number of CMs, and increased the cross-sectional area of CMs. However, the glucocorticoid receptor antagonist had no effect on CM proliferation. Agonists of adenylate cyclase and protein kinase A (PKA) inhibited CM proliferation, while PKA antagonists or knockdown of PKA alleviated the inhibitory effect of GCs on CM proliferation. GCs and the activation of the cyclic adenosine monophosphate (cAMP)/PKA signaling pathway facilitated yes-associated protein (YAP) phosphorylation in mouse CMs and promoted YAP protein translocation from the nucleus to the cytoplasm. Meanwhile, blocking the cAMP/PKA signaling pathway partially blocked the effect of GCs on YAP protein phosphorylation and YAP protein translocation. Thus, our findings suggest that GCs suppress mouse CM proliferation in vitro and in vivo, through a mechanism that involves targeting the cAMP-PKA-YAP signaling pathway., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The gene encoding the cAMP-dependent protein kinase (PKA) catalytic subunit-like protein PKAC1 from the Venezuelan TeAp-N/D1 strain of Trypanosoma equiperdum was cloned, and the recombinant TeqPKAC1 protein was overexpressed in bacteria. A major polypeptide with an apparent molecular mass of ∼38 kDa was detected by SDS-polyacrylamide gel electrophoresis, and immunoblotting using antibodies against the human PKA catalytic subunit α. Unfortunately, most of the expressed TeqPKAC1 was highly insoluble. Polypeptides of 36-38 kDa and 45-50 kDa were predominantly seen by immunoblotting in the bacterial particulate and cytosolic fractions, respectively. Since the incorporation of either 4% Triton X-100 or 3% sarkosyl or a mixture of 10 mM MgCl 2 and 1 mM ATP (MgATP) improved the solubilization of TeqPKAC1, we used a combination of Triton X-100, sarkosyl and MgATP to solubilize the recombinant protein. TeqPKAC1 was purified by first reconstituting a hybrid holoenzyme between the recombinant protein and a mammalian poly-His-tagged PKA regulatory subunit that was immobilized on a Ni 2+ -chelating affinity resin, and then by eluting TeqPKAC1 using cAMP. TeqPKAC1 was functional given that it was capable of phosphorylating PKA catalytic subunit substrates, such as kemptide (LRRASLG), histone type II-AS, and the peptide SP20 (TTYADFIASGRTGRRNSIHD), and was inhibited by the peptide IP20 (TTYADFIASGRTGRRNAIHD), which contains the inhibitory motif of the PKA-specific heat-stable inhibitor PKI-α. Optimal enzymatic activity was obtained at 37 °C and pH 8.0-9.0; and the order of effectiveness of nucleotide triphosphates and divalent cations was ATP » GTP ≅ ITP and Mg 2+  ≅ Mn 2+  ≅ Fe 2+ » Ca 2+  ≅ Zn 2 , respectively., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cyclic AMP is a ubiquitous second messenger used to transduce intracellular signals from a variety of Gs-coupled receptors. Compartmentalisation of protein intermediates within the cAMP signaling pathway underpins receptor-specific responses. The cAMP effector proteins protein-kinase A and EPAC are found in complexes that also contain phosphodiesterases whose presence ensures a coordinated cellular response to receptor activation events. Popeye domain containing (POPDC) proteins are the most recent class of cAMP effectors to be identified and have crucial roles in cardiac pacemaking and conduction. We report the first observation that POPDC proteins exist in complexes with members of the PDE4 family in cardiac myocytes. We show that POPDC1 preferentially binds the PDE4A sub-family via a specificity motif in the PDE4 UCR1 region and that PDE4s bind to the Popeye domain of POPDC1 in a region known to be susceptible to a mutation that causes human disease. Using a cell-permeable disruptor peptide that displaces the POPDC1-PDE4 complex we show that PDE4 activity localized to POPDC1 modulates cycle length of spontaneous Ca 2+ transients firing in intact mouse sinoatrial nodes., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Beyond natural stimuli such as growth factors and stresses, the ability to experimentally modulate at will the levels or activity of specific intracellular signaling molecule(s) in specified cells within a tissue can be a powerful tool for uncovering new regulation and tissue behaviors. Here we perturb the levels of cAMP within specific cells of an epithelial monolayer to probe the time-dynamic behavior of cell-cell communication protocols implemented by the cAMP/PKA pathway and its coupling to the ERK pathway. The time-dependent ERK responses we observe in the perturbed cells for spatially uniform cAMP perturbations (all cells) can be very different from those due to spatially localized perturbations (a few cells). Through a combination of pharmacological and genetic perturbations, signal analysis, and computational modeling, we infer how intracellular regulation and regulated cell-cell coupling each impact the intracellular ERK response in single cells. Our approach reveals how a dynamic gap junction state helps sculpt the intracellular ERK response over time in locally perturbed cells., Competing Interests: The authors have declared that no competing interests exist.

Longitudinal bone growth is achieved by a tightly controlled process termed endochondral bone formation. C-type natriuretic peptide (CNP) stimulates endochondral bone formation through binding to its specific receptor, guanylyl cyclase (GC)-B. However, CNP/GC-B signaling dynamics in different stages of endochondral bone formation have not been fully clarified, especially in terms of the interaction between the cyclic guanine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) pathways. Here, we demonstrated that CNP activates the cAMP/protein kinase A (PKA) pathway and that this activation contributed to the elongation of the hypertrophic zone in the growth plate. Cells of the chondrogenic line ATDC5 were transfected with Förster resonance energy transfer (FRET)-based cGMP and PKA biosensors. Dual-FRET imaging revealed that CNP increased intracellular cGMP levels and PKA activities in chondrocytes. Further, CNP-induced PKA activation was enhanced following differentiation of ATDC5 cells. Live imaging of the fetal growth plate of transgenic mice, expressing a FRET biosensor for PKA, PKAchu mice, showed that CNP predominantly activates the PKA in the hypertrophic chondrocytes. Additionally, histological analysis of the growth plate of PKAchu mice demonstrated that CNP increased the length of the growth plate, but coadministration of a PKA inhibitor, H89, inhibited the growth-promoting effect of CNP only in the hypertrophic zone. In summary, we revealed that CNP-induced cGMP elevation activated the cAMP/PKA pathway, and clarified that this PKA activation contributed to the bone growth-promoting effect of CNP in hypertrophic chondrocytes. These results provide insights regarding the cross-talk between cGMP and cAMP signaling in endochondral bone formation and in the physiological role of the CNP/GC-B system., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society.)

Kinase activity in signaling networks frequently depends on regulatory subunits that can both inhibit activity by interacting with the catalytic subunits and target the kinase to distinct molecular partners and subcellular compartments. Here, using a new synthetic molecular interaction system, we show that translocation of a regulatory subunit of the protein kinase A (PKA-R) to the plasma membrane has a paradoxical effect on the membrane kinase activity. It can both enhance it at lower translocation levels, even in the absence of signaling inputs, and inhibit it at higher translocation levels, suggesting its role as a linker that can both couple and decouple signaling processes in a concentration-dependent manner. We further demonstrate that superposition of gradients of PKA-R abundance across single cells can control the directionality of cell migration, reversing it at high enough input levels. Thus, complex in vivo patterns of PKA-R localization can drive complex phenotypes, including cell migration., Competing Interests: RL, BL, TK, AL No competing interests declared, (© 2022, LaCroix et al.)