Adenylyl Cyclase Activity Research Articles

Repurposing FDA-Approved Drugs Against Potential Drug Targets Involved in Brain Inflammation Contributing to Alzheimer’s Disease

Alzheimer’s disease is a neurodegenerative disease that continues to have a rising number of cases. While extensive research has been conducted on Alzheimer’s disease in the last few decades, only a few drugs have been approved by the FDA for its treatment, and even fewer aim to be curative rather than manage symptoms. There remains an urgent need to understand disease pathogenesis, as well as identify new targets for further drug discovery. Alzheimer’s disease (AD) is known to stem from the build-up of amyloid beta (Aβ) plaques, as well as tangles of tau proteins. Furthermore, inflammation in the brain is known to arise from the degeneration of tissue and the build-up of insoluble material. Therefore, there is a potential link between the pathology of AD and inflammation in the brain, especially as the disease progresses to later stages, where neuronal death and degeneration levels are higher. Proteins that are relevant to both brain inflammation and AD, thus, make ideal potential targets for therapeutics; however, the proteins need to be evaluated to determine which targets would be ideal for potential drug therapeutic treatments, or ‘druggable’ targets. Druggability analysis was conducted using two structure-based methods (i.e., drug-like density analysis and SiteMap), as well as a sequence-based approach, SPIDER. The most druggable targets were then evaluated using single-nucleus sequencing data for their clinical relevance to inflammation in AD. For each of the top five targets, small molecule docking was used to evaluate which FDA approved drugs were able to bind with the chosen proteins. The top targets included DRD2 (inhibits adenylyl cyclase activity), C9 (binds with C5B8 to form the membrane attack complex), C4b (binds with C2a to form C3 convertase), C5AR1 (a GPCR that binds C5a), and GABA-A-R (the GPCR involved in inhibiting neurotransmission). Each target had multiple potential inhibitors from the FDA-approved drug list with decent binding infinities. Among these inhibitors, two drugs were found to be top inhibitors for more than one protein target. They were C15H14N2O2 and v316 (paracetamol), originally used to treat pain/inflammation for cataracts and relieve headaches/fever, respectively. These results provide the groundwork for further experimental investigations or clinical trials.

Intracellular cAMP signaling-induced Ca2+ influx mediated by calcium homeostasis modulator 1 (CALHM1) in human odontoblasts.

In odontoblasts, intracellular Ca2+ signaling plays key roles in reactionary dentin formation and generation of dentinal pain. Odontoblasts also express several Gs protein-coupled receptors that promote production of cyclic AMP (cAMP). However, the crosstalk between intracellular cAMP and Ca2+ signaling, as well as the role of cAMP in the cellular functions of odontoblasts, remains unclear. In this study, we measured intracellular cAMP levels and intracellular free Ca2+ concentration ([Ca2+]i). We also investigated the effect of intracellular cAMP on mineralization by the odontoblasts. In the presence of extracellular Ca2+, the application of forskolin (adenylyl cyclase activator) or isoproterenol (Gs protein-coupled beta-2 adrenergic receptor agonist) increased intracellular cAMP levels and [Ca2+]i in odontoblasts. The [Ca2+]i increases could not be observed by removing extracellular Ca2+, indicating that cAMP is capable to activate Ca2+ entry. Forskolin-induced [Ca2+]i increase was inhibited by a protein kinase A inhibitor in odontoblasts. The [Ca2+]i increase was sensitive to Gd3+, 2APB, or Zn2+ but not verapamil, ML218, or La3+. In immunofluorescence analyses, odontoblasts were immunopositive for calcium homeostasis modulator 1 (CALHM1), which was found close to ionotropic ATP receptor subtype, P2X3 receptors. When CALHM1 was knocked down, forskolin-induced [Ca2+]i increase was suppressed. Alizarin red and von Kossa staining showed that forskolin decreased mineralization. These findings suggest that activation of adenylyl cyclase elicited increases in the intracellular cAMP level and Ca2+ influx via protein kinase A activation in odontoblasts. Subsequent cAMP-dependent Ca2+ influx was mediated by CALHM1 in odontoblasts. In addition, the intracellular cAMP signaling pathway in odontoblasts negatively mediated dentinogenesis.

Modulation of left ventricular hypertrophy in spontaneously hypertensive rats by acetylcholinesterase and ACE inhibitors: physiological, biochemical, and proteomic studies

BackgroundThe consequences at the molecular level and the mechanisms involved in a possible cardioprotective effect of antihypertensive treatment are not yet fully understood. Here, the efficacy of pyridostigmine (PYR) and trandolapril (TRA) as antihypertensive and antihypertrophic agents was investigated and compared in hypertensive SHR and normotensive WKY rats. In parallel, we investigated the effects of these drugs on myocardial β-adrenergic and cholinergic signaling pathways and protein expression profiles.MethodsAge-matched male SHR and WKY rats were chronically (8 weeks) treated with PYR or TRA in drinking water. Blood pressure (BP) and heart rate (HR) were monitored telemetrically prior to tissue sampling for biochemical analysis. Baroreceptor reflex sensitivity (BRS) and methylatropine HR response as a measure of vagal tone were evaluated in separate groups of animals.ResultsPYR slightly lowered BP and HR in SHR rats during the dark phase of the day, while TRA effectively reduced BP during the light and dark phases without affecting HR. PYR enhanced BRS and improved vagal tone. There were no significant alterations in myocardial β-adrenergic and cholinergic signaling, with the exception of decreased forskolin-stimulated adenylyl cyclase (AC) activity in SHR rats, which was restored by TRA. Proteomic analysis revealed numerous differences induced by both treatments. Notable were changes in TGFβ-related signaling pathways as well as proteins involved in modifying hemodynamic parameters and cardiac hypertrophy.ConclusionsPYR is able to slightly decrease BP and HR in SHR rats but effectively increase BRS through vagal potentiation. The specific differences in protein expression profiles in rat myocardium induced by treatment with PYR and TRA reflect different mechanisms of action of these two agents at the molecular level.

Cantharidin and sodium fluoride attenuate the negative inotropic effect of the A1-adenosine receptor agonist N6-(R)-phenylisopropyl adenosine in isolated human atria.

Cantharidin and sodium fluoride inhibit the activity of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A) and increase the force of contraction in human atrial preparations. R-phenylisopropyl adenosine (R-PIA) acts as an agonist at A1-adenosine receptors. R-PIA exerts a negative inotropic effect on human atria. The effect of R-PIA-and its various manifestations-are currently explained as a function of the inhibition of sarcolemmal adenylyl cyclase activity and/or opening of sarcolemmal potassium channels. We hypothesise that cantharidin and sodium fluoride may attenuate the negative inotropic effect of R-PIA. During open heart surgery, trabeculae carneae from the right atrium were obtained for human atrial preparations (HAPs). These trabeculae were mounted in organ baths and electrically stimulated at 1Hz. Furthermore, we studied isolated electrically stimulated left atrial (LA) preparations from female wild-type mice (CD1). The force of contraction was recorded under isometric conditions. R-PIA (1µM) exerted a rapid negative inotropic effect in the HAPs and mice LA preparations. These negative inotropic effects of R-PIA were attenuated by pre-incubation for 30min with 100-µM cantharidin in HAPs, but not in mice LA preparations. Adenosine signals via A1 receptors in a species-specific pathway in mammalian atria. We postulate that R-PIA, at least in part, exerts a negative inotropic effect via activation of serine/threonine phosphatases in the human atrium.

CFTR Inhibitors Display Antiviral Activity against Herpes Simplex Virus.

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

Extracellular cAMP elicits contraction of rat vas deferens: Involvement of ecto-5′-nucleotidase and adenosine A1 receptors

AimsIt is well established that intracellular cAMP contributes to the relaxation of vas deferens smooth muscle. In many tissues, intracellular cAMP is actively transported to the extracellular space, where it exerts regulatory functions, via its metabolite adenosine. These actions take place through the cAMP conversion to adenosine by ectoenzymes, a process called “extracellular cAMP-adenosine pathway”. Herein, we investigated whether, in addition to ATP, extracellular cAMP might be an alternative source of adenosine, influencing the contraction of vas deferens smooth muscle. Main methodsThe effects of cAMP, 8-Br-cAMP and adenosine were analyzed in the isometric contractions of rat vas deferens. cAMP efflux was analyzed by measuring extracellular cAMP levels after exposure of vas deferens segments to isoproterenol and forskolin in the presence or absence of MK-571, an inhibitor of MRP/ABCC transporters. Key findingsWhile 8-Br-cAMP, a cell-permeable cAMP analog, induced relaxation of KCl-precontracted vas deferens, the non-permeant cAMP increased the KCl-induced contractile response, which was mimicked by adenosine, but prevented by inhibitors of ecto-5′-nucleotidase or A1 receptors. Our results also showed that isoproterenol and forskolin increases cAMP efflux via an MRP/ABCC transporter-dependent mechanism, since it is inhibited by MK-571. SignificanceOur data show that activation of β-adrenoceptors and adenylyl cyclase increases cAMP efflux from vas deferens tissue, which modulates the vas deferens contractile response via activation of adenosine A1 receptors. Assuming that inhibition of vas deferens contractility has been proposed as a strategy for male contraception, the extracellular cAMP-adenosine pathway emerges as a potential pharmacological target that should be considered in studies of male fertility.

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.

Abstract Tu077: Targeting β-adrenergic receptor resensitization attenuates cardiac dysfunction

β-adrenergic receptor (βAR) is a key regulator of cardiac function. Agonist activation of βAR leads to phosphorylation-mediated desensitization resulting in βAR trafficking to endosomes. Endosomal βARs undergo resensitization through protein phosphatase 2A (PP2A)-mediated dephosphorylation. We have shown that stimulation of βARs activates phosphoinositide 3-kinase γ (PI3Kγ) that phosphorylates endogenous inhibitor of PP2A (I2PP2A) promoting robust binding to PP2A resulting in PP2A inhibition. βAR dysfunction due to desensitization is considered as a classical hallmark of heart failure, while less is known about resensitization. Plasma membrane and endosomal fractions were isolated from non-failing and failing patient samples to determine whether βAR resensitization is altered in human heart failure. Endosomes from heart failure samples showed significant accumulation of phosphorylated β2ARs which was associated with reduced adenylyl cyclase and βAR-associated PP2A activity compared to non-failing showing that resensitization is inhibited in human heart failure. Since PP2A activity is inhibited by robust binding of I2PP2A to PP2A, we hypothesized that disrupting I2PP2A interaction with PP2A would unlock PP2A inhibition normalizing βAR resensitization. In silico modeling and biochemical studies mapped the PP2A interaction with I2PP2A to the C-terminal region of PP2A (PP2Act). Agonist isoproterenol (ISO) treatment of cells expressing PP2Act showed significant reduction in β2AR phosphorylation along with increased cAMP generation and normalized PP2A activity showing preserved βAR resensitization. While expression of PP2A mutant with deletion in the PP2Act (ΔPP2Act) did not preserve βAR resensitization. Challenging mice with cardiomyocyte-specific expression of PP2Act to ISO showed preserved cardiac function providing insights that targeting resensitization could potentially be a novel therapeutic strategy. Key words: Resensitization, I2PP2A, PP2A, Cardiac hypertrophy

Induction of long-term hyperexcitability by memory-related cAMP signaling in isolated nociceptor cell bodies.

Persistent hyperactivity of nociceptors is known to contribute significantly to long-lasting sensitization and ongoing pain in many clinical conditions. It is often assumed that nociceptor hyperactivity is mainly driven by continuing stimulation from inflammatory mediators. We have tested an additional possibility: that persistent increases in excitability promoting hyperactivity can be induced by a prototypical cellular signaling pathway long known to induce late-phase long-term potentiation (LTP) of synapses in brain regions involved in memory formation. This cAMP-PKA-CREB-gene transcription-protein synthesis pathway was tested using whole-cell current clamp methods on small dissociated sensory neurons (primarily nociceptors) from dorsal root ganglia (DRGs) excised from previously uninjured ("naïve") rats. Six-hour treatment with the specific Gαs-coupled 5-HT4 receptor agonist, prucalopride, or with the adenylyl cyclase activator, forskolin, induced long-term hyperexcitability (LTH) in DRG neurons that manifested 12-24 hours later as action potential (AP) discharge (ongoing activity, OA) during artificial depolarization to -45 mV, a membrane potential that is normally subthreshold for AP generation. Prucalopride treatment also induced significant long-lasting depolarization of resting membrane potential (from -69 to -66 mV), enhanced depolarizing spontaneous fluctuations (DSFs) of membrane potential, and indications of reduced AP threshold and rheobase. LTH was prevented by co-treatment of prucalopride with inhibitors of PKA, CREB, gene transcription, and protein synthesis. As in the induction of synaptic memory, many other cellular signals are likely to be involved. However, the discovery that this prototypical memory induction pathway can induce nociceptor LTH, along with reports that cAMP signaling and CREB activity in DRGs can induce hyperalgesic priming, suggest that early, temporary, cAMP-induced transcriptional and translational mechanisms can induce nociceptor LTH that might last for long periods. An interesting possibility is that these mechanisms can also be reactivated by re-exposure to inflammatory mediators such as serotonin during subsequent challenges to bodily integrity, "reconsolidating" the cellular memory and thereby extending the duration of persistent nociceptor hyperexcitability.

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

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. 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. 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. This study suggests that forskolin can contribute potentially to improve chronic olfactory dysfunction post COVID-19. DFM-IRB00012367-23-10-001.

A liver immune rheostat regulates CD8 T cell immunity in chronic HBV infection

Chronic hepatitis B virus (HBV) infection affects 300 million patients worldwide1,2, in whom virus-specific CD8 T cells by still ill-defined mechanisms lose their function and cannot eliminate HBV-infected hepatocytes3–7. Here we demonstrate that a liver immune rheostat renders virus-specific CD8 T cells refractory to activation and leads to their loss of effector functions. In preclinical models of persistent infection with hepatotropic viruses such as HBV, dysfunctional virus-specific CXCR6+ CD8 T cells accumulated in the liver and, as a characteristic hallmark, showed enhanced transcriptional activity of cAMP-responsive element modulator (CREM) distinct from T cell exhaustion. In patients with chronic hepatitis B, circulating and intrahepatic HBV-specific CXCR6+ CD8 T cells with enhanced CREM expression and transcriptional activity were detected at a frequency of 12–22% of HBV-specific CD8 T cells. Knocking out the inhibitory CREM/ICER isoform in T cells, however, failed to rescue T cell immunity. This indicates that CREM activity was a consequence, rather than the cause, of loss in T cell function, further supported by the observation of enhanced phosphorylation of protein kinase A (PKA) which is upstream of CREM. Indeed, we found that enhanced cAMP–PKA-signalling from increased T cell adenylyl cyclase activity augmented CREM activity and curbed T cell activation and effector function in persistent hepatic infection. Mechanistically, CD8 T cells recognizing their antigen on hepatocytes established close and extensive contact with liver sinusoidal endothelial cells, thereby enhancing adenylyl cyclase–cAMP–PKA signalling in T cells. In these hepatic CD8 T cells, which recognize their antigen on hepatocytes, phosphorylation of key signalling kinases of the T cell receptor signalling pathway was impaired, which rendered them refractory to activation. Thus, close contact with liver sinusoidal endothelial cells curbs the activation and effector function of HBV-specific CD8 T cells that target hepatocytes expressing viral antigens by means of the adenylyl cyclase–cAMP–PKA axis in an immune rheostat-like fashion.

Induction of long-term hyperexcitability by memory-related cAMP signaling in isolated nociceptor cell bodies

Persistent hyperactivity of nociceptors is known to contribute significantly to long-lasting sensitization and ongoing pain in many clinical conditions. It is often assumed that nociceptor hyperactivity is mainly driven by continuing stimulation from inflammatory mediators. We have tested an additional possibility: that persistent increases in excitability promoting hyperactivity can be induced by a prototypical cellular signaling pathway long known to induce late-phase long-term potentiation (LTP) of synapses in brain regions involved in memory formation. This cAMP-PKA-CREB-gene transcription-protein synthesis pathway was tested using whole-cell current clamp methods on small dissociated sensory neurons (primarily nociceptors) from dorsal root ganglia (DRGs) excised from previously uninjured (“naïve”) male rats. Six-hour treatment with the specific Gαs-coupled 5-HT4 receptor agonist, prucalopride, or with the adenylyl cyclase activator forskolin induced long-term hyperexcitability (LTH) in DRG neurons that manifested 12–24 h later as action potential (AP) discharge (ongoing activity, OA) during artificial depolarization to −45 mV, a membrane potential that is normally subthreshold for AP generation. Prucalopride treatment also induced significant long-lasting depolarization of resting membrane potential (from −69 to −66 mV), enhanced depolarizing spontaneous fluctuations (DSFs) of membrane potential, and produced trends for reduced AP threshold and rheobase. LTH was prevented by co-treatment of prucalopride with inhibitors of PKA, CREB, gene transcription, or protein synthesis. As in the induction of synaptic memory, many other cellular signals are likely to be involved. However, the discovery that this prototypical memory induction pathway can induce nociceptor LTH, along with reports that cAMP signaling and CREB activity in DRGs can induce hyperalgesic priming, suggest that early, temporary, cAMP-induced transcriptional and translational mechanisms can induce nociceptor LTH that might last for long periods. The present results also raise the question of whether reactivation of primed signaling mechanisms by re-exposure to inflammatory mediators linked to cAMP synthesis during subsequent challenges to bodily integrity can “reconsolidate” nociceptor memory, extending the duration of persistent hyperexcitability.

Dual role of valosin-containing protein (VCP/p97) in mouse sperm during capacitation.

Valosin-containing protein (VCP; aka p97), a member of the AAA (ATPases Associated with various cellular Activities) family, has been associated with a wide range of cellular functions. While previous evidence has shown its presence in mammalian sperm, our study unveils its function in mouse sperm. Notably, we found that mouse VCP does not undergo tyrosine phosphorylation during capacitation and exhibits distinct localization patterns. In the sperm head, it resides within the equatorial segment and, following acrosomal exocytosis, it is released and cleaved. In the flagellum, VCP is observed in the principal and midpiece. Furthermore, our research highlights a unique role for VCP in the cAMP/PKA pathway during capacitation. Pharmacological inhibition of sperm VCP led to reduced intracellular cAMP levels that resulted in decreased phosphorylation in PKA substrates and tyrosine residues and diminished fertilization competence. Our results show that in mouse sperm, VCP plays a pivotal role in regulating cAMP production, probably by the modulation of soluble adenylyl cyclase activity.

(007) TESTOSTERONE POSITIVELY REGULATES ADENOSINE-INDUCED RELAXATION IN THE VAGINA: AN EXPERIMENTAL STUDY IN RATS

Abstract Introduction It has been previously demonstrated that testosterone (T) has a pivotal role in controlling vaginal relaxation through the related molecular machinery, i.e., improving the nitric oxide (NO)/protein kinase G (PKG)/cyclic guanosine monophosphate (cGMP) vaginal signaling and maintaining the integrity of the muscular wall. Adenosine (ADO) is a purinergic neuromodulator that, in the male, shows a positive effect on penile erection by controlling smooth muscle relaxation and local blood flow increase. However, the role, if any, of T on the ADO-induced relaxation signaling is yet to be investigated. Objective The aim of this study was to evaluate the effect of sex steroids on ADO-mediated vaginal relaxation using a validated animal model of ovariectomized (OVX) and sex steroid supplemented Sprague-Dawley rats. Methods Subgroups of OVX rats were treated with 17β-estradiol (E2; 10 mg/kg/die), testosterone (T; 30 mg/kg/week), or testosterone and letrozole (T+L, 2.5 mg/kg/die) for 6 weeks. The experimental groups were compared with a group of intact rats. In vitro contractility studies were performed on noradrenaline (NA) pre-contracted distal vaginal strips, isolated from each experimental group, stimulated with increasing doses of ADO (1 μM - 1 mM), with or without NO-synthase inhibitor L-NAME (100 μM) pretreatment, or treated with ADORA2A selective agonist CGS21680 (10 Pm-100 μM). Messenger ribonucleic acid (mRNA) isolated from vaginal tissue was analyzed by semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction. Results The results showed that OVX resulted in a decreased responsiveness to ADO, completely restored by T supplementation, with or without letrozole. In contrast, E2 did not affect the hyporesponsiveness to ADO in OVX rats. Likewise, ADO induced dose-dependent relaxation was significantly reduced by in vitro L-NAME (100 μM) only in the OVX animals treated with T or T+L. Accordingly, vagina expressed all ADO receptors (ADORA1, ADORA2A, ADORA2B, ADORA3) mRNAs, with pro-relaxant ADORA2A (ADO receptor coupled to adenylyl cyclase activation) being significantly modulated by hormonal treatments: OVX significantly reduced ADORA2A mRNA expression, an effect counteracted by either T or T+L administration, but not by E2. These data are supported by the observed T-induced upregulation of cyclic adenosine monophosphate (cAMP)-dependent genes, where T, but not E2, treatment upregulated the mRNA expression of the ubiquitous isoform of ADCY (ADCY4), protein kinase A (PKA) catalytic subunit b (PKAcb), PKA regulatory subunit 2b (PKAr2b) and cAMP responsive element modulator (CREM). Finally, as further demonstration of the functional activity of ADO signaling in distal vagina, stimulation by increasing doses of CGS21680, a selective agonist of ADORA2A receptor, enhanced the relaxant effect induced by T and T+L treatments but not by E2. Conclusions Our data demonstrated for the first time a novel ADO-mediated relaxant mechanism in vagina and suggested that T positively modulates the responsiveness to ADO stimulation, thus confirming this process as a major player in the relaxant mechanism regulation of muscle contractility in vagina. Disclosure No.

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.

Primary Infection and Supernatants Generated by Pseudomonas aeruginosa ExoY Have Lasting Effects on Cyclic Nucleotide Signaling in Pulmonary Endothelial Cells

Rationale: Pseudomonas aeruginosa is an opportunistic pathogen and a common cause of hospital-acquire pneumonia. This gram-negative bacterium has an arsenal of virulence factors including the type three secretion system (TTSS), which encodes for an injection type needle and exotoxins. One of the four exotoxins, ExoY, is a promiscuous nucleotidylyl cyclase that utilizes F-actin as a cofactor to generate both canonical and non-canonical cyclic nucleotides (cAMP, cGMP and cCMP and cUMP respectively). While it is well known that activation of endogenous adenylyl and guanylyl cyclase leads to intracellular increases in cAMP and cGMP and release into the extracellular space, whether ExoY generated cyclic nucleotides are also effluxed out of the cell into the extracellular space is not known. In addition, it is unclear whether presence of the bacteria themselves affects endogenous cAMP signaling. Further, infection with P. aeruginosa expressing ExoY generates virulent cytotoxic amyloids from lung endothelium; however, whether these cytotoxic amyloids disrupt agonist-induced endogenous cAMP generation is unknown. Thus, we tested the hypothesis that during primary infection, cAMP and cGMP are effluxed into the extracellular space, while the primary infection itself and sterile supernatants from the primary infection attenuate isoproterenol induce increases in cAMP in pulmonary microvascular endothelial cells (PMVECs). Methods: During primary infection, PMVECs were inoculated with PBS (vehicle control) or 3 different strains of P. aeruginosa at an moi 20:1. Strains ExoY+ and ExoYK81M have a fully functional TTSS and express either catalytically active or inactive ExoY, respectively, while a third strain, ExoYTM, is unable to translocate ExoY into eukaryotic cells. During the last 15 minutes of the 4-hour infection, cells were stimulated with vehicle or isoproterenol (1 μM) and rolipram (10 μM) and cAMP and cGMP measured in the lysate and media. In separate experiments, supernatants from a primary infection were generated using the same three strains of bacteria at moi 20:1 and 4 hours after inoculation, the supernatants collected and sterilized using a 0.2 μm filter. Sterilized supernatants were added to naïve PMVECs and during the last 15 minutes of the 4-hour inoculation, cells stimulated with vehicle or isoproterenol (1 μM) and rolipram (10 μM) and cAMP and cGMP measured in the lysate and media. Results: Cyclic AMP and cGMP were elevated in both the lysate and media of PMVECs following 4-hour infection with ExoY+ and the total isoproterenol and rolipram-stimulated cAMP response was attenuated by the presence of bacteria, ExoYK81M and ExoYTM, and further blunted with ExoY+ infection. Further, inoculation with ExoYK81M and ExoYTM supernatants attenuated the total isoproterenol and rolipram-stimulated cAMP response, which was further blunted by ExoY+ supernatants. Conclusions: Overall, we demonstrate PMVECs release a large cAMP and cGMP pool into the extracellular space and both primary infection and supernatants derived from the primary infection attenuate agonist induced elevations in cAMP. HL121513, HL66299. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PKA inhibition is a central step in D,L-methadone-induced ER Ca2+ release and subsequent apoptosis in acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is a hematologic cancer that mostly affects children. It accounts for over a quarter of ALL pediatric cancers, causing most of the cancer death among children. Previously, we demonstrated that D,L-methadone causes ALL cell apoptosis via μ-opioid receptor 1 (OPRM1)-triggered ER Ca2+ release and decrease in Ca2+ efflux, elevating [Ca2+]i. However, the precise mechanism by which D,L-methadone induces ER Ca2+ release remains to be defined. Here, we show that in ALL cells, D,L-methadone-induced ER Ca2+ release is blocked by inhibition of Gαi, but not Gβϒ, indicating that the process is dependent on Gαi. Activation of adenylyl cyclase (AC) with forskolin or treatment with 8-CPT-cAMP blocks D,L-methadone-induced ER Ca2+ release, indicating that the latter results from Gαi-dependent downregulation of AC and cAMP. The 14-22 amide (myr) PKA inhibitor alone elicits ER Ca2+ release, and subsequent treatment with D,L-methadone does not cause additional ER Ca2+ release, indicating that PKA inhibition is a key step in D,L-methadone-induced ER Ca2+ release and can bypass the D,L-methadone-OPRM1-AC-cAMP step. This is consistent with the decrease in PKA-dependent (i) inhibitory PLCβ3 Ser1105 phosphorylation that leads to PLCβ3 activation and ER Ca2+ release, and (ii) BAD Ser118 phosphorylation, which together ultimately result in caspase activation and apoptosis. Thus, our findings indicate that D,L-methadone-induced ER Ca2+ release and subsequent apoptosis in ALL cells is mediated by Gαi-dependent downregulation of the AC-cAMP-PKA-PLCβ3/BAD pathway. The fact that 14-22 amide (myr) alone effectively kills ALL cells suggests that PKA may be targeted for ALL therapy.

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.

GLP-1 in the Hypothalamic Paraventricular Nucleus Promotes Sympathetic Activation and Hypertension.

Glucagon-like peptide-1 (GLP-1) and its analogs are widely used for diabetes treatment. The paraventricular nucleus (PVN) is crucial for regulating cardiovascular activity. This study aims to determine the roles of GLP-1 and its receptors (GLP-1R) in the PVN in regulating sympathetic outflow and blood pressure. Experiments were carried out in male normotensive rats and spontaneously hypertensive rats (SHR). Renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded. GLP-1 and GLP-1R expressions were present in the PVN. PVN microinjection of GLP-1R agonist recombinant human GLP-1 (rhGLP-1) or EX-4 increased RSNA and MAP, which were prevented by GLP-1R antagonist exendin 9-39 (EX9-39) or GLP-1R antagonist 1, superoxide scavenger tempol, antioxidant N-acetylcysteine, NADPH oxidase (NOX) inhibitor apocynin, adenylyl cyclase (AC) inhibitor SQ22536 or protein kinase A (PKA) inhibitor H89. PVN microinjection of rhGLP-1 increased superoxide production, NADPH oxidase activity, cAMP level, AC, and PKA activity, which were prevented by SQ22536 or H89. GLP-1 and GLP-1R were upregulated in the PVN of SHR. PVN microinjection of GLP-1 agonist increased RSNA and MAP in both WKY and SHR, but GLP-1 antagonists caused greater effects in reducing RSNA and MAP in SHR than in WKY. The increased superoxide production and NADPH oxidase activity in the PVN of SHR were augmented by GLP-1R agonists but attenuated by GLP-1R antagonists. These results indicate that activation of GLP-1R in the PVN increased sympathetic outflow and blood pressure via cAMP-PKA-mediated NADPH oxidase activation and subsequent superoxide production. GLP-1 and GLP-1R upregulation in the PVN partially contributes to sympathetic overactivity and hypertension.

Abstract 4712: GPCR panel establishment and application in drug discovery

Abstract G protein-coupled receptors (GPCRS) are the largest family of membrane proteins of cell surface receptors. It can be activated by various stimuli and play an important role in various physiological and pathological processes such as cell growth, proliferation, differentiation, metabolism, and homeostasis. Abnormal regulation of GPCR is associated with various human diseases, such as metabolic diseases, cardiovascular diseases, and eye diseases. G protein contains three subunits: α, β, and γ, which form a trimer. When extracellular ligands are bound to GPCRs which activate G protein and GTP phosphorylates Gα subunits, leading to the dissociation of Gα subunits from the trimer and dissociation from the β and γ subunits. In addition, Gα different subtypes of Gαs, Gαi and Gαq have different biological effects after activation, Gas-coupled receptors activate adenylyl cyclase (AC) to increase cAMP. In contrast, Gαi-coupled receptors activated adenylyl cyclase (AC) to inhibit cAMP. Gαq coupled receptors promote phospholipase C (PLC) to produce IP3, and IP3 can increase intracellular Ca2+ concentration. GPCR Panel With high general availability, which provides over 150 GPCR targets, covering wide families like 5-Hydroxytryptamine, adrenoceptors, Acetylcholine, dopamine, glucagon, and opioid receptors and having the selectivity of multiple species. It can conduct similar GPCR poly-link interaction and screening, used to study new GPCR drug development technology, detect the expression properties of GPCR receptors, reveal new GPCR structure and function, and screen GPCR drugs. We also constructed different assay platforms, including cAMP assay, Ca2+ flux assay, IP3 assay, β-arrestin recruitment assay and reporter assay to detect the different second messengers produced by G protein submits and GPCR-mediated multiple signaling pathways. This research constructed stable cell lines expressing GPCRs and provided related assays, which could be used to detect compounds' effects on GPCR targets successfully. In addition, the GPCR panel was applicable to both activating and inhibitory models, and the establishment of this platform provides a powerful tool for drug development of GPCR-related targets. Citation Format: Xiaoyan Wang, Ning Sui, Dandan Hu, Ziwei Zhang, Tiejun Bing. GPCR panel establishment and application in drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4712.