Spatial organization of adenylyl cyclase and its impact on dopamine signaling in neurons.

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.

Moonlighting Crypto-Enzymes and Domains as Ancient and Versatile Signaling Devices.

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

Adenylate cyclase 1 knockdown attenuates pirarubicin-induced cardiotoxicity.

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

A molecular mechanism to diversify Ca2+ signaling downstream of Gs protein-coupled receptors.

A long-held tenet in inositol-lipid signaling is that cleavage of membrane phosphoinositides by phospholipase Cß (PLCß) isozymes to increase cytosolic Ca2+ in living cells is exclusive to Gq- and Gi-sensitive G protein-coupled receptors (GPCRs). Here we extend this central tenet and show that Gs-GPCRs also partake in inositol-lipid signaling and thereby increase cytosolic Ca2+. By combining CRISPR/Cas9 genome editing to delete Gαs, the adenylyl cyclase isoforms 3 and 6, or the PLCß1-4 isozymes, with pharmacological and genetic inhibition of Gq and G11, we pin down Gs-derived Gßγ as driver of a PLCß2/3-mediated cytosolic Ca2+ release module. This module does not require but crosstalks with Gαs-dependent cAMP, demands Gαq to release PLCß3 autoinhibition, but becomes Gq-independent with mutational disruption of the PLCß3 autoinhibited state. Our findings uncover the key steps of a previously unappreciated mechanism utilized by mammalian cells to finetune their calcium signaling regulation through Gs-GPCRs.

Cyclic AMP is a global virulence regulator governing inter and intrabacterial signalling in Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic nosocomial pathogen with high morbidity and mortality rates. Current treatment options for this pathogen are limited due to its increasing resistance to last-resort antibiotics. Despite A. baumannii's leading position in the World Health Organisations priority pathogens list, little is known about its virulence regulation. Through a high-throughput screening approach to identify novel biofilm regulators, we identified a previously uncharacterised predicted adenylate cyclase (AC), CavA, as a central regulator of this phenotype. cAMP is a crucial mediator of various aspects of bacterial physiology in other species but information about its role in A. baumannii is limited. We confirm that CavA AC is functional and synthesizes cAMP in A. baumannii. Using dRNA-seq, we verify that CavA is a negative biofilm formation regulator affecting Csu pili and exopolysaccharide production. We demonstrate for the first time that in A. baumannii, cAMP is atop of a hierarchical signalling cascade controlling inter- and intrabacterial signalling by modulating quorum sensing and cyclic di-GMP systems, ultimately governing virulence in vivo and adaptive antibiotic resistance. In contrast to the well-established paradigm in other bacteria where cAMP and cyclic di-GMP levels are inversely regulated, we uncover that the levels of these second messengers are directly proportional in A. baumannii. Overall, this study uncovers the central role of CavA and cAMP in the pathogenic success of A. baumannii and highlights this signalling cascade as a high potential target for novel therapeutic development.

Identification of diagnostic biomarkers and immune cell profiles associated with COPD integrated bioinformatics and machine learning.

This retrospective transcriptomic study leveraged bioinformatics and machine learning algorithms to identify novel gene biomarkers and explore immune cell infiltration profiles associated with chronic obstructive pulmonary disease (COPD). Utilizing an integrated analysis of metadata encompassing six gene expression omnibus (GEO) microarray datasets, 987 differentially expressed genes were identified. Further gene ontology and pathway enrichment analyses revealed the enrichment of these genes across various biological processes and pathways. Moreover, a systematic integration of two machine learning algorithms along with pathway-gene correlations identified six candidate biomarkers, which were validated in a separate cohort comprising six additional microarray datasets, ultimately identifying ADD3 and GNAS as diagnostic biomarkers for COPD. Subsequently, the diagnostic efficacy of ADD3 and GNAS was assessed, and the impact of their expression levels on overall survival was further evaluated and quantified in the validation cohort. Examination of immune cell subtype infiltration found increased proportions of cytotoxic CD8+ T cells, resting and activated NK cells, along with decreased M0 and M2 macrophages, in COPD versus control samples. Correlation analyses also uncovered significant associations between ADD3 and GNAS expression and infiltration of various immune cell types. In conclusion, this study elucidates crucial COPD diagnostic biomarkers and immune cell profiles which may illuminate the immunopathological drivers of COPD progression, representing personalized therapeutic targets warranting further investigation.

Effect of Nitrosyl Iron Complex with 3,4-Dichlorothiophenolyls on the Level of Cyclic Nucleotide In Vitro.

The effect of a promising NO donor, a binuclear nitrosyl iron complex (NIC) with 3,4-dichlorothiophenolyls [Fe2(SC6H3Cl2)2(NO)4], on the adenylate cyclase and soluble guanylate cyclase enzymatic systems was studied. In in vitro experiments, this complex increased the concentration of important secondary messengers, such as cAMP and cGMP. An increase of their level by 2.4 and 4.5 times, respectively, was detected at NIC concentration of 0.1 mM. The ligand of the complex, 3,4-dichlorothiophenol, produced a less pronounced effect on adenylate cyclase. It was shown that the effect of this complex on the activity of soluble guanylate cyclase was comparable to the effect of anionic nitrosyl complex with thiosulfate ligands that exhibits vasodilating and cardioprotective properties.

Voltage-gated calcium channels act upstream of adenylyl cyclase Ac78C to promote timely initiation of dendrite regeneration.

Most neurons are not replaced after injury and thus possess robust intrinsic mechanisms for repair after damage. Axon injury triggers a calcium wave, and calcium and cAMP can augment axon regeneration. In comparison to axon regeneration, dendrite regeneration is poorly understood. To test whether calcium and cAMP might also be involved in dendrite injury signaling, we tracked the responses of Drosophila dendritic arborization neurons to laser severing of axons and dendrites. We found that calcium and subsequently cAMP accumulate in the cell body after both dendrite and axon injury. Two voltage-gated calcium channels (VGCCs), L-Type and T-Type, are required for the calcium influx in response to dendrite injury and play a role in rapid initiation of dendrite regeneration. The AC8 family adenylyl cyclase, Ac78C, is required for cAMP production after dendrite injury and timely initiation of regeneration. Injury-induced cAMP production is sensitive to VGCC reduction, placing calcium upstream of cAMP generation. We propose that two VGCCs initiate global calcium influx in response to dendrite injury followed by production of cAMP by Ac78C. This signaling pathway promotes timely initiation of dendrite regrowth several hours after dendrite damage.

KCTD proteins regulate morphine dependence via heterologous sensitization of adenylyl cyclase 1 in mice.

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

Soluble adenylyl cyclase is an acid-base sensor in rainbow trout red blood cells that regulates intracellular pH and haemoglobin-oxygen binding.

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

Adenylyl cyclase 2 expression and function in neurological diseases.

Adenylyl cyclases (Adcys) catalyze the formation of cAMP, a secondary messenger essential for cell survival and neurotransmission pathways in the CNS. Adcy2, one of ten Adcy isoforms, is highly expressed in the CNS. Abnormal Adcy2 expression and mutations have been reported in various neurological disorders in both rodents and humans. However, due to the lack of genetic tools, loss-of-function studies of Adcy2 are scarce. In this review, we summarize recent findings on Adcy2 expression and function in neurological diseases. Specifically, we first introduce the biochemistry, structure, and function of Adcy2 briefly. Next, the expression and association of Adcy2 in human patients and rodent models of neurodegenerative diseases (Alzheimer's disease and Parkinson's disease), psychiatric disorders (Tourette syndrome, schizophrenia, and bipolar disorder), and other neurological conditions (stress-associated disorders, stroke, epilepsy, and Lesch-Nyhan Syndrome) are elaborated. Furthermore, we discuss the pros and cons of current studies as well as key questions that need to be answered in the future. We hope to provide a focused review on Adcy2 that promotes future research in the field.

Effect of Xanthohumol, a Bioactive Natural Compound from Hops, on Adenosine Pathway in Rat C6 Glioma and Human SH-SY5Y Neuroblastoma Cell Lines.

Xanthohumol (Xn) is an antioxidant flavonoid mainly extracted from hops (Humulus lupulus), one of the main ingredients of beer. As with other bioactive compounds, their therapeutic potential against different diseases has been tested, one of which is Alzheimer's disease (AD). Adenosine is a neuromodulatory nucleoside that acts through four different G protein-coupled receptors: A1 and A3, which inhibit the adenylyl cyclases (AC) pathway, and A2A and A2B, which stimulate this activity, causing either a decrease or an increase, respectively, in the release of excitatory neurotransmitters such as glutamate. This adenosinergic pathway, which is altered in AD, could be involved in the excitotoxicity process. Therefore, the aim of this work is to describe the effect of Xn on the adenosinergic pathway using cell lines. For this purpose, two different cellular models, rat glioma C6 and human neuroblastoma SH-SY5Y, were exposed to a non-cytotoxic 10 µM Xn concentration. Adenosine A1 and A2A, receptor levels, and activities related to the adenosine pathway, such as adenylate cyclase, protein kinase A, and 5'-nucleotidase, were analyzed. The adenosine A1 receptor was significantly increased after Xn exposure, while no changes in A2A receptor membrane levels or AC activity were reported. Regarding 5'-nucleotidases, modulation of their activity by Xn was noted since CD73, the extracellular membrane attached to 5'-nucleotidase, was significantly decreased in the C6 cell line. In conclusion, here we describe a novel pathway in which the bioactive flavonoid Xn could have potentially beneficial effects on AD as it increases membrane A1 receptors while modulating enzymes related to the adenosine pathway in cell cultures.

Cordycepin Triphosphate as a Potential Modulator of Cellular Plasticity in Cancer via cAMP-Dependent Pathways: An In Silico Approach.

Cordycepin, or 3'-deoxyadenosine, is an adenosine analog with a broad spectrum of biological activity. The key structural difference between cordycepin and adenosine lies in the absence of a hydroxyl group at the 3' position of the ribose ring. Upon administration, cordycepin can undergo an enzymatic transformation in specific tissues, forming cordycepin triphosphate. In this study, we conducted a comprehensive analysis of the structural features of cordycepin and its derivatives, contrasting them with endogenous purine-based metabolites using chemoinformatics and bioinformatics tools in addition to molecular dynamics simulations. We tested the hypothesis that cordycepin triphosphate could bind to the active site of the adenylate cyclase enzyme. The outcomes of our molecular dynamics simulations revealed scores that are comparable to, and superior to, those of adenosine triphosphate (ATP), the endogenous ligand. This interaction could reduce the production of cyclic adenosine monophosphate (cAMP) by acting as a pseudo-ATP that lacks a hydroxyl group at the 3' position, essential to carry out nucleotide cyclization. We discuss the implications in the context of the plasticity of cancer and other cells within the tumor microenvironment, such as cancer-associated fibroblast, endothelial, and immune cells. This interaction could awaken antitumor immunity by preventing phenotypic changes in the immune cells driven by sustained cAMP signaling. The last could be an unreported molecular mechanism that helps to explain more details about cordycepin's mechanism of action.

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.

Forskolin is an effective therapeutic small molecule for the treatment of hypertrophic cardiomyopathy through ADCY6/cAMP/PKA pathway.

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 (Myh6R404Q and Tnnt2R109Q) 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.

Enhanced long-term potentiation in the anterior cingulate cortex of tree shrew.

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

Alleviation of migraine related pain and anxiety by inhibiting calcium-stimulating AC1-dependent CGRP in the insula of adult rats.

BACKGROUND: Recent animal and clinical findings consistently highlight the critical role of calcitonin gene-related peptide (CGRP) in chronic migraine (CM) and related emotional responses. CGRP antibodies and receptor antagonists have been approved for CM treatment. However, the underlying CGRP-related signaling pathways in the pain-related cortex remain poorly understood. METHODS: The SD rats were used to establish the CM model by dural infusions of inflammatory soup. Periorbital mechanical thresholds were assessed using von-Frey filaments, and anxiety-like behaviors were observed via open field and elevated plus maze tests. Expression of c-Fos, CGRP and NMDA GluN2B receptors was detected using immunofluorescence and western blotting analyses. The excitatory synaptic transmission was detected by whole-cell patch-clamp recording. A human-used adenylate cyclase 1 (AC1) inhibitor, hNB001, was applied via insula stereotaxic and intraperitoneal injections in CM rats. RESULTS: The insular cortex (IC) was activated in the migraine model rats. Glutamate-mediated excitatory transmission and NMDA GluN2B receptors in the IC were potentiated. CGRP levels in the IC significantly increased during nociceptive and anxiety-like activities. Locally applied hNB001 in the IC or intraperitoneally alleviated periorbital mechanical thresholds and anxiety behaviors in migraine rats. Furthermore, CGRP expression in the IC decreased after the hNB001 application. CONCLUSIONS: Our study indicated that AC1-dependent IC plasticity contributes to migraine and AC1 may be a promising target for treating migraine in the future.

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.

A CatSper-Uninvolved Mechanism to Induce Forward Sperm Motility in the Internal Fertilization.

Sperm-specific cation channel (CatSper), sperm-specific Na + /H + exchanger (sNHE), and soluble adenylyl cyclase (sAC) are necessary in the signaling pathways to control sperm motility in many animals, whereas some animals have lost some or all of them. In the present study, we examined CatSper-uninvolved signaling for vigorous undulation of the undulating membrane that is attached to the sperm tail and gives thrust for forward motility in the internally fertilizing newt Cynops pyrrhogaster. Reverse-transcription PCR failed to detect sNHE in the newt sperm. However, the pH of sperm cytoplasm was raised under a high extracellular pH equivalent to that of egg jelly, where sperm motility is initiated by sperm motility-initiating substance (SMIS). Carbonic anhydrase XII/ XVI and SLC4A4/8 were suggested to be present in the sperm, and transported bicarbonates raised the intracellular pH. In egg jelly extract that contained SMIS, the anion transporter inhibitor DIDS weakened the undulation of the undulating membrane, while bicarbonates enhanced it. The cyclic AMP concentration was found to increase in sperm cytoplasm in the egg-jelly extract. An inhibitor of sAC (KH7) weakened the undulation of the undulating membrane, and dibutyryl cyclic AMP blocked the inhibitory effect. Inhibitor of transmembrane AC (DDA) limitedly affected the undulation. The undulation was weakened by an inhibitor of protein kinase A (H89), and by an inhibitor of transient receptor potential (TRP) channels (RN1747). Our results support the conclusions that the high pH of the egg jelly triggers a signaling pathway through sAC, PKA, and TRP channels, and coacts with SMIS to induce forward sperm motility.

Testosterone Enhances KV Currents and Airway Smooth Muscle Relaxation Induced by ATP and UTP through P2Y4 Receptors and Adenylyl Cyclase Pathway.

Numerous studies suggest the involvement of adenosine-5'-triphosphate (ATP) and similar nucleotides in the pathophysiology of asthma. Androgens, such as testosterone (TES), are proposed to alleviate asthma symptoms in young men. ATP and uridine-5'-triphosphate (UTP) relax the airway smooth muscle (ASM) via purinergic P2Y2 and P2Y4 receptors and K+ channel opening. We previously demonstrated that TES increased the expression of voltage-dependent K+ (KV) channels in ASM. This study investigates how TES may potentiate ASM relaxation induced by ATP and UTP. Tracheal tissues treated with or without TES (control group) from young male guinea pigs were used. In organ baths, tracheas exposed to TES (40 nM for 48 h) showed enhanced ATP- and UTP-evoked relaxation. Tetraethylammonium, a K+ channel blocker, annulled this effect. Patch-clamp experiments in tracheal myocytes showed that TES also increased ATP- and UTP-induced K+ currents, and this effect was abolished with flutamide (an androgen receptor antagonist). KV channels were involved in this phenomenon, which was demonstrated by inhibition with 4-aminopyridine. RB2 (an antagonist of almost all P2Y receptors except for P2Y2), as well as N-ethylmaleimide and SQ 22,536 (inhibitors of G proteins and adenylyl cyclase, respectively), attenuated the enhancement of the K+ currents induced by TES. Immunofluorescence and immunohistochemistry studies revealed that TES did not modify the expression of P2Y4 receptors or COX-1 and COX-2, while we have demonstrated that this androgen augmented the expression of KV1.2 and KV1.5 channels in ASM. Thus, TES leads to the upregulation of P2Y4 signaling and KV channels in guinea pig ASM, enhancing ATP and UTP relaxation responses, which likely limits the severity of bronchospasm in young males.