Hormonal and Allosteric Regulation of the Luteinizing Hormone/Chorionic Gonadotropin Receptor.

Luteinizing hormone (LH) and human chorionic gonadotropin (CG), like follicle-stimulating hormone, are the most important regulators of the reproductive system. They exert their effect on the cell through the LH/CG receptor (LHCGR), which belongs to the family of G protein-coupled receptors. Binding to gonadotropin induces the interaction of LHCGR with various types of heterotrimeric G proteins (Gs, Gq/11, Gi) and ß-arrestins, which leads to stimulation (Gs) or inhibition (Gi) of cyclic adenosine monophosphate-dependent cascades, activation of the phospholipase pathway (Gq/11), and also to the formation of signalosomes that mediate the stimulation of mitogen-activated protein kinases (ß-arrestins). The efficiency and selectivity of activation of intracellular cascades by different gonadotropins varies, which is due to differences in their interaction with the ligand-binding site of LHCGR. Gonadotropin signaling largely depends on the status of N- and O-glycosylation of LH and CG, on the formation of homo- and heterodimeric receptor complexes, on the cell-specific microenvironment of LHCGR and the presence of autoantibodies to it, and allosteric mechanisms are important in the implementation of these influences, which is due to the multiplicity of allosteric sites in different loci of the LHCGR. The development of low-molecular-weight allosteric regulators of LHCGR with different profiles of pharmacological activity, which can be used in medicine for the correction of reproductive disorders and in assisted reproductive technologies, is promising. These and other issues regarding the hormonal and allosteric regulation of LHCGR are summarized and discussed in this review.

Mechanism of gabapentinoid potentiation of opioid effects on cyclic AMP signaling in neuropathic pain.

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.

Adenylyl Cyclase Isoform 6 in the Pulmonary Artery Is Inhibited by Hypoxia via Cysteine Nitrosylation.

Persistent pulmonary hypertension of the newborn (PPHN) is a hypoxic disorder of pulmonary vascular relaxation, mediated in part by adenylyl cyclase (AC). Neonatal pulmonary arteries (PA) express mainly AC6 isoform, followed by AC3, 7 and 9. AC6 expression is upregulated in hypoxia. We reported AC enzyme inhibition due to S-nitrosylation in PPHN PA, and in PA myocytes exposed to hypoxia. We hypothesize that hypoxia promotes cysteine thiol nitrosylation of AC6, impairing cAMP production. HEK293T cells stably expressing AC isoforms (AC3, 5, 6, 7, 9), or cysteine-to-alanine mutants AC6_C1004A, AC6_C1145A or AC6_C447A were cultured in normoxia (21% O2) or hypoxia (10% O2) for 72 hours, or challenged with nitroso donor S-nitrosocysteine (CysNO). AC activity was determined by real-time live-cell cAMP measurement (cADDis assay) or terbium-norfloxacin AC catalytic assay, with or without challenge by allosteric agonist forskolin; protein S-nitrosylation detected by biotin switch method and quantified by affinity precipitation. Only AC6 catalytic activity is inhibited in hypoxia or by S-nitrosylating agent, in presence or absence of forskolin; impaired cAMP production in hypoxia correlates with increased cysteine nitrosylation of AC6. Selective AC6 inhibition in pulmonary artery myocytes extinguishes AC sensitivity to inhibition by hypoxia. Alanine substitution of C1004, but not of other cysteines, decreases S-nitrosylation of AC6. AC activity is diminished in AC6_C1004A compared to AC6 wild type. Substitution of C1004 also extinguishes the inhibition of AC6 by hypoxia. We conclude AC6 is uniquely S-nitrosylated in hypoxia, inhibiting its activity and cAMP generation. We speculate that S-nitrosylation at C1004 may inhibit AC6 interaction with Gαs, playing a role in PPHN pathophysiology.

Activation of IP3R in atrial cardiomyocytes leads to generation of cytosolic cAMP.

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.

Efficacy of forskolin as a promising therapy for chronic olfactory dysfunction post COVID-19.

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.

Integrated bioinformatics investigation of adenylyl cyclase family co-expression network in bladder cancer followed by preliminary validation of member 2 (ADCY2) in tumorigenesis and prognosis.

Background: The adenylyl cyclase (ADCY) gene family encodes enzymes responsible for the synthesis of cyclic adenosine monophosphate (cAMP) from adenosine triphosphate (ATP), which comprises nine transmembrane isoforms (ADCYs 1-9). Although ADCYs correlate with intracellular signalling and tumorigenesis in different malignancies, their roles in bladder cancer remain unclear. Methods: Utilizing the bladder urothelial carcinoma (BLCA) dataset from The Cancer Genome Atlas (TCGA), we employed the R package 'limma' to identify differential genes. Subsequent correlation analysis with corresponding clinical data was conducted. Prognostic significance of ADCY family genes was assessed through survival analysis. Univariate and multivariate Cox regression determined ADCY2 as a potential independent risk factor for BLCA. Validation was performed using immunohistochemistry results from independent cohorts. Additionally, we delved into the mechanism of genetic variations, methylation modifications, and signalling pathways of ADCY family genes. Evaluation of their role in the immune microenvironment was achieved through R packages single-sample gene set enrichment analysis (ssGSEA), CIBERPORT, and ESTIMATE. Results: Cases of bladder cancer were retrieved from TCGA, and the transcriptionally differentially expressed members of ADCY were identified (members 2, 4, and 5). Genomic alteration, epigenomic modification, clinicopathological characteristics and clinical survival were systematically investigated. A co-expression network was established based on the intersection of correlated genes, which was centred around ADCY2, ADCY4, and ADCY5. Enrichment analysis revealed that correlated genes were involved in epithelial-mesenchymal transition (EMT). The ADCY2 was selected as the most representative biomarker for prognosis in bladder cancer. Bladder tumour with higher ADCY2 expression had higher prognostic risk and worse survival outcomes. Moreover, ADCY2 was correlated with classic immune checkpoints, and a better responsiveness to immunotherapy was exhibited in high-expression subsets. To ameliorate universality of the conclusion, our study also included several real-world cohorts into the preliminary validation, using datasets from the Gene Expression Omnibus (GEO; GSE13507), tissue microarray (TMA) with 80 bladder cancer inclusion and clinical trial IMvigor210, which were associated with immunotherapy sensitivity, prognosis, and common biomarker presentation. Conclusions: Our study reveals that ADCY family has prognostic value in patients with bladder cancer; the ADCY2 is a prominent prognostic biomarker. The bioinformatics analyses and validation provide direction for further functional and mechanistic studies on the screened members of ADCY family.

Adenylyl Cyclase in Ocular Health and Disease: A Comprehensive Review.

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.

Inhibition of calcium-stimulated adenylyl cyclase subtype 1 (AC1) for the treatment of pain and anxiety symptoms in Parkinson's disease mice model.

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.

Administration of Bicarbonate Protects Mitochondria, Rescues Retinal Ganglion Cells, and Ameliorates Visual Dysfunction Caused by Oxidative Stress.

Oxidative stress is a key factor causing mitochondrial dysfunction and retinal ganglion cell (RGC) death in glaucomatous neurodegeneration. The cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway is involved in mitochondrial protection, promoting RGC survival. Soluble adenylyl cyclase (sAC) is a key regulator of the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway, which is known to protect mitochondria and promote RGC survival. However, the precise molecular mechanisms connecting the sAC-mediated signaling pathway with mitochondrial protection in RGCs against oxidative stress are not well characterized. Here, we demonstrate that sAC plays a critical role in protecting RGC mitochondria from oxidative stress. Using mouse models of oxidative stress induced by ischemic injury and paraquat administration, we found that administration of bicarbonate, as an activator of sAC, protected RGCs, blocked AMP-activated protein kinase activation, inhibited glial activation, and improved visual function. Moreover, we found that this is the result of preserving mitochondrial dynamics (fusion and fission), promoting mitochondrial bioenergetics and biogenesis, and preventing metabolic stress and apoptotic cell death. Notably, the administration of bicarbonate ameliorated mitochondrial dysfunction in RGCs by enhancing mitochondrial biogenesis, preserving mitochondrial structure, and increasing ATP production in oxidatively stressed RGCs. These findings suggest that activating sAC enhances the mitochondrial structure and function in RGCs to counter oxidative stress, consequently promoting RGC protection. We propose that modulation of the sAC-mediated signaling pathway has therapeutic potential acting on RGC mitochondria for treating glaucoma and other retinal diseases.

To each its own: Mechanisms of cross-talk between GPI biosynthesis and cAMP-PKA signaling in Candida albicans versus Saccharomyces cerevisiae.

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.

Recruitment of cortical silent responses by forskolin in the anterior cingulate cortex of adult mice.

Recent studies using different experimental approaches demonstrate that silent synapses may exist in the adult cortex including the sensory cortex and anterior cingulate cortex (ACC). The postsynaptic form of long-term potentiation (LTP) in the ACC recruits some of these silent synapses and the activity of calcium-stimulated adenylyl cyclases (ACs) is required for such recruitment. It is unknown if the chemical activation of ACs may recruit silent synapses. In this study, we found that activation of ACs contributed to synaptic potentiation in the ACC of adult mice. Forskolin, a selective activator of ACs, recruited silent responses in the ACC of adult mice. The recruitment was long-lasting. Interestingly, the effect of forskolin was not universal, some silent synapses did not undergo potentiation or recruitment. These findings suggest that these adult cortical synapses are not homogenous. The application of a selective calcium-permeable AMPA receptor inhibitor 1-naphthyl acetyl spermine (NASPM) reversed the potentiation and the recruitment of silent responses, indicating that the AMPA receptor is required. Our results strongly suggest that the AC-dependent postsynaptic AMPA receptor contributes to the recruitment of silent responses at cortical LTP.

The A-kinase anchoring protein Yotiao decrease the ER calcium content by inhibiting the store operated calcium entry.

The meticulous regulation of ER calcium (Ca2+) homeostasis is indispensable for the proper functioning of numerous cellular processes. Disrupted ER Ca2+ balance is implicated in diverse diseases, underscoring the need for a systematic exploration of its regulatory factors in cells. Our recent genomic-scale screen identified a scaffolding protein A-kinase anchoring protein 9 (AKAP9) as a regulator of ER Ca2+ levels, but the underlying molecular mechanisms remain elusive. Here, we reveal that Yotiao, the smallest splicing variant of AKAP9 decreased ER Ca2+ content in animal cells. Additional testing using a combination of Yotiao truncations, knock-out cells and pharmacological tools revealed that, Yotiao does not require most of its interactors, including type 1 inositol 1,4,5-trisphosphate receptors (IP3R1), protein kinase A (PKA), protein phosphatase 1 (PP1), adenylyl cyclase type 2 (AC2) and so on, to reduce ER Ca2+ levels. However, adenylyl cyclase type 9 (AC9), which is known to increases its cAMP generation upon interaction with Yotiao for the modulation of potassium channels, plays an essential role for Yotiao's ER-Ca2+-lowering effect. Mechanistically, Yotiao may work through AC9 to act on Orai1-C terminus and suppress store operated Ca2+ entry, resulting in reduced ER Ca2+ levels. These findings not only enhance our comprehension of the interplay between Yotiao and AC9 but also contribute to a more intricate understanding of the finely tuned mechanisms governing ER Ca2+ homeostasis.

The uniqueness of on-demand male contraception.

Because nearly half of pregnancies worldwide are unintended, available contraceptive methods are inadequate. Moreover, due to the striking imbalance between contraceptive options available for men compared to the myriad of options available to women, there is an urgent need for new methods of contraception for men. This review summarizes ongoing efforts to develop male contraceptives highlighting the unique aspects particular to on-demand male contraception, where a man takes a contraceptive only when and as often as needed.

The role of soluble adenylyl cyclase in sensing and regulating intracellular pH.

Soluble adenylyl cyclase (sAC) differs from transmembrane adenylyl cyclases (tmAC) in many aspects. In particular, the activity of sAC is not regulated by G-proteins but by the prevailing bicarbonate concentrations inside cells. Therefore, sAC serves as an exquisite intracellular pH sensor, with the capacity to translate pH changes into the regulation of localization and/or activity of cellular proteins involved in pH homeostasis. In this review, we provide an overview of literature describing the regulation of sAC activity by bicarbonate, pinpointing the importance of compartmentalization of intracellular cAMP signaling cascades. In addition, examples of processes involving proton and bicarbonate transport in different cell types, in which sAC plays an important regulatory role, were described in detail.

Erythrocytes membrane fluidity changes induced by adenylyl cyclase cascade activation: study using fluorescence recovery after photobleaching.

In this study, fluorescence recovery after photobleaching (FRAP) experiments were performed on RBC labeled by lipophilic fluorescent dye CM-DiI to evaluate the role of adenylyl cyclase cascade activation in changes of lateral diffusion of erythrocytes membrane lipids. Stimulation of adrenergic receptors with epinephrine (adrenaline) or metaproterenol led to the significant acceleration of the FRAP recovery, thus indicating an elevated membrane fluidity. The effect of the stimulation of protein kinase A with membrane-permeable analog of cAMP followed the same trend but was less significant. The observed effects are assumed to be driven by increased mobility of phospholipids resulting from the weakened interaction between the intermembrane proteins and RBC cytoskeleton due to activation of adenylyl cyclase signaling cascade.

Pharmacological potential of cyclic nucleotide signaling in immunity.

Cyclic nucleotides are important signaling molecules that play many critical physiological roles including controlling cell fate and development, regulation of metabolic processes, and responding to changes in the environment. Cyclic nucleotides are also pivotal regulators in immune signaling, orchestrating intricate processes that maintain homeostasis and defend against pathogenic threats. This review provides a comprehensive examination of the pharmacological potential of cyclic nucleotide signaling pathways within the realm of immunity. Beginning with an overview of the fundamental roles of cAMP and cGMP as ubiquitous second messengers, this review delves into the complexities of their involvement in immune responses. Special attention is given to the challenges associated with modulating these signaling pathways for therapeutic purposes, emphasizing the necessity for achieving cell-type specificity to avert unintended consequences. A major focus of the review is on the recent paradigm-shifting discoveries regarding specialized cyclic nucleotide signals in the innate immune system, notably the cGAS-STING pathway. The significance of cyclic dinucleotides, exemplified by 2'3'-cGAMP, in controlling immune responses against pathogens and cancer, is explored. The evolutionarily conserved nature of cyclic dinucleotides as antiviral agents, spanning across diverse organisms, underscores their potential as targets for innovative immunotherapies. Findings from the last several years have revealed a striking diversity of novel bacterial cyclic nucleotide second messengers which are involved in antiviral responses. Knowledge of the existence and precise identity of these molecules coupled with accurate descriptions of their associated immune defense pathways will be essential to the future development of novel antibacterial therapeutic strategies. The insights presented herein may help researchers navigate the evolving landscape of immunopharmacology as it pertains to cyclic nucleotides and point toward new avenues or lines of thinking about development of therapeutics against the pathways they regulate.

Adenylyl cyclase isoforms 5 and 6 in the cardiovascular system: complex regulation and divergent roles.

Adenylyl cyclases (ACs) are crucial effector enzymes that transduce divergent signals from upstream receptor pathways and are responsible for catalyzing the conversion of ATP to cAMP. The ten AC isoforms are categorized into four main groups; the class III or calcium-inhibited family of ACs comprises AC5 and AC6. These enzymes are very closely related in structure and have a paucity of selective activators or inhibitors, making it difficult to distinguish them experimentally. AC5 and AC6 are highly expressed in the heart and vasculature, as well as the spinal cord and brain; AC6 is also abundant in the lungs, kidney, and liver. However, while AC5 and AC6 have similar expression patterns with some redundant functions, they have distinct physiological roles due to differing regulation and cAMP signaling compartmentation. AC5 is critical in cardiac and vascular function; AC6 is a key effector of vasodilatory pathways in vascular myocytes and is enriched in fetal/neonatal tissues. Expression of both AC5 and AC6 decreases in heart failure; however, AC5 disruption is cardio-protective, while overexpression of AC6 rescues cardiac function in cardiac injury. This is a comprehensive review of the complex regulation of AC5 and AC6 in the cardiovascular system, highlighting overexpression and knockout studies as well as transgenic models illuminating each enzyme and focusing on post-translational modifications that regulate their cellular localization and biological functions. We also describe pharmacological challenges in the design of isoform-selective activators or inhibitors for AC5 and AC6, which may be relevant to developing new therapeutic approaches for several cardiovascular diseases.

The cAMP signaling module regulates sperm motility in the liverwort Marchantia polymorpha.

Adenosine 3',5'-cyclic monophosphate (cAMP) is a universal signaling molecule that acts as a second messenger in various organisms. It is well established that cAMP plays essential roles across the tree of life, although the function of cAMP in land plants has long been debated. We previously identified the enzyme with both adenylyl cyclase (AC) and cAMP phosphodiesterase (PDE) activity as the cAMP-synthesis/hydrolysis enzyme COMBINED AC with PDE (CAPE) in the liverwort Marchantia polymorpha. CAPE is conserved in streptophytes that reproduce with motile sperm; however, the precise function of CAPE is not yet known. In this study, we demonstrate that the loss of function of CAPE in M. polymorpha led to male infertility due to impaired sperm flagellar motility. We also found that two genes encoding the regulatory subunits of cAMP-dependent protein kinase (PKA-R) were also involved in sperm motility. Based on these findings, it is evident that CAPE and PKA-Rs act as a cAMP signaling module that regulates sperm motility in M. polymorpha. Therefore, our results have shed light on the function of cAMP signaling and sperm motility regulators in land plants. This study suggests that cAMP signaling plays a common role in plant and animal sperm motility.

Two-Photon FRET/FLIM Imaging of Cerebral Neurons.

Two-photon FRET (Förster resonance energy transfer) and FLIM (fluorescence lifetime imaging microscopy) enable the detection of FRET changes of fluorescence reporters in deep brain tissues, which provide a valuable approach for monitoring target molecular dynamics and functions. Here, we describe two-photon FRET and FLIM imaging techniques that allow us to visualize endogenous and optogenetically induced cAMP dynamics in living neurons with genetically engineered FRET-based cAMP reporters.

RelA-mediated signaling connects adaptation to chronic cardiomyocyte stress with myocardial and systemic inflammation in the ADCY8 model of accelerated aging.

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.