On-demand male contraception via acute inhibition of soluble adenylyl cyclase.

Nearly half of all pregnancies are unintended; thus, existing family planning options are inadequate. For men, the only choices are condoms and vasectomy, and most current efforts to develop new contraceptives for men impact sperm development, meaning that contraception requires months of continuous pretreatment. Here, we provide proof-of-concept for an innovative strategy for on-demand contraception, where a man would take a birth control pill shortly before sex, only as needed. Soluble adenylyl cyclase (sAC) is essential for sperm motility and maturation. We show a single dose of a safe, acutely-acting sAC inhibitor with long residence time renders male mice temporarily infertile. Mice exhibit normal mating behavior, and full fertility returns the next day. These studies define sAC inhibitors as leads for on-demand contraceptives for men, and they provide in vivo proof-of-concept for previously untested paradigms in contraception; on-demand contraception after just a single dose and pharmacological contraception for men.

PTH1R translocation to primary cilia in mechanically-stimulated ostecytes prevents osteoclast formation via regulation of CXCL5 and IL-6 secretion.

Osteocytes respond to mechanical forces controlling osteoblast and osteoclast function. Mechanical stimulation decreases osteocyte apoptosis and promotes bone formation. Primary cilia have been described as potential mechanosensors in bone cells. Certain osteogenic responses induced by fluid flow (FF) in vitro are decreased by primary cilia inhibition in MLO-Y4 osteocytes. The parathyroid hormone (PTH) receptor type 1 (PTH1R) modulates osteoblast, osteoclast, and osteocyte effects upon activation by PTH or PTH-related protein (PTHrP) in osteoblastic cells. Moreover, some actions of PTH1R seem to be triggered directly by mechanical stimulation. We hypothesize that PTH1R forms a signaling complex in the primary cilium that is essential for mechanotransduction in osteocytes and affects osteocyte-osteoclast communication. MLO-Y4 osteocytes were stimulated by FF or PTHrP (1-37). PTH1R and primary cilia signaling were abrogated using PTH1R or primary cilia specific siRNAs or inhibitors, respectively. Conditioned media obtained from mechanically- or PTHrP-stimulated MLO-Y4 cells inhibited the migration of preosteoclastic cells and osteoclast differentiation. Redistribution of PTH1R along the entire cilium was observed in mechanically stimulated MLO-Y4 osteocytic cells. Preincubation of MLO-Y4 cells with the Gli-1 antagonist, the adenylate cyclase inhibitor (SQ22536), or with the phospholipase C inhibitor (U73122), affected the migration of osteoclast precursors and osteoclastogenesis. Proteomic analysis and neutralizing experiments showed that FF and PTH1R activation control osteoclast function through the modulation of C-X-C Motif Chemokine Ligand 5 (CXCL5) and interleukin-6 (IL-6) secretion in osteocytes. These novel findings indicate that both primary cilium and PTH1R are necessary in osteocytes for proper communication with osteoclasts and show that mechanical stimulation inhibits osteoclast recruitment and differentiation through CXCL5, while PTH1R activation regulate these processes via IL-6.

Halogen-Dance-Based Synthesis of Phosphonomethoxyethyl (PME) Substituted 2-Aminothiazoles as Potent Inhibitors of Bacterial Adenylate Cyclases.

A series of acyclic nucleoside phosphonates (ANPs) was designed as inhibitors of bacterial adenylate cyclases (ACs), where adenine was replaced with 2-amino-4-arylthiazoles. The target compounds were prepared using the halogen dance reaction. Final AC inhibitors were evaluated in cell-based assays (prodrugs) and cell-free assays (phosphono diphosphates). Novel ANPs were potent inhibitors of adenylate cyclase toxin (ACT) from Bordetella pertussis and edema factor (EF) from Bacillus anthracis, with substantial selectivity over mammalian enzymes AC1, AC2, and AC5. Six of the new ANPs were more potent or equipotent ACT inhibitors (IC50 =9-18 nM), and one of them was more potent EF inhibitor (IC50 =12 nM), compared to adefovir diphosphate (PMEApp) with IC50 =18 nM for ACT and IC50 =36 nM for EF. Thus, these compounds represent the most potent ACT/EF inhibitors based on ANPs reported to date. The potency of the phosphonodiamidates to inhibit ACT activity in J774A.1 macrophage cells was somewhat weaker, where the most potent derivative had IC50 =490 nM compared to IC50 =150 nM of the analogous adefovir phosphonodiamidate. The results suggest that more efficient type of phosphonate prodrugs would be desirable to increase concentrations of the ANP-based active species in the cells in order to proceed with the development of ANPs as potential antitoxin therapeutics.

Therapeutic melanoma inhibition by local micelle-mediated cyclic nucleotide repression.

The acidic tumor microenvironment in melanoma drives immune evasion by up-regulating cyclic adenosine monophosphate (cAMP) in tumor-infiltrating monocytes. Here we show that the release of non-toxic concentrations of an adenylate cyclase (AC) inhibitor from poly(sarcosine)-block-poly(L-glutamic acid γ-benzyl ester) (polypept(o)id) copolymer micelles restores antitumor immunity. In combination with selective, non-therapeutic regulatory T cell depletion, AC inhibitor micelles achieve a complete remission of established B16-F10-OVA tumors. Single-cell sequencing of melanoma-infiltrating immune cells shows that AC inhibitor micelles reduce the number of anti-inflammatory myeloid cells and checkpoint receptor expression on T cells. AC inhibitor micelles thus represent an immunotherapeutic measure to counteract melanoma immune escape.

Soluble adenylyl cyclase inhibition prevents human sperm functions essential for fertilization.

Soluble adenylyl cyclase (sAC: ADCY10) has been genetically confirmed to be essential for male fertility in mice and humans. In mice, ex vivo studies of dormant, caudal epididymal sperm demonstrated that sAC is required for initiating capacitation and activating motility. We now use an improved sAC inhibitor, TDI-10229, for a comprehensive analysis of sAC function in mouse and human sperm. In contrast to caudal epididymal mouse sperm, human sperm are collected post-ejaculation, after sAC activity has already been stimulated. In addition to preventing the capacitation-induced stimulation of sAC and protein kinase A activities, tyrosine phosphorylation, alkalinization, beat frequency and acrosome reaction in dormant mouse sperm, sAC inhibitors interrupt each of these capacitation-induced changes in ejaculated human sperm. Furthermore, we show for the first time that sAC is required during acrosomal exocytosis in mouse and human sperm. These data define sAC inhibitors as candidates for non-hormonal, on-demand contraceptives suitable for delivery via intravaginal devices in women.

Pharmacological inhibition of mitochondrial soluble adenylyl cyclase in astrocytes causes activation of AMP-activated protein kinase and induces breakdown of glycogen.

Mobilization of astrocyte glycogen is key for processes such as synaptic plasticity and memory formation but the link between neuronal activity and glycogen breakdown is not fully known. Activation of cytosolic soluble adenylyl cyclase (sAC) in astrocytes has been suggested to link neuronal depolarization and glycogen breakdown partly based on experiments employing pharmacological inhibition of sAC. However, several studies have revealed that sAC located within mitochondria is a central regulator of respiration and oxidative phosphorylation. Thus, pharmacological sAC inhibition is likely to affect both cytosolic and mitochondrial sAC and if bioenergetic readouts are studied, the observed effects are likely to stem from inhibition of mitochondrial rather than cytosolic sAC. Here, we report that a pharmacologically induced inhibition of sAC activity lowers mitochondrial respiration, induces phosphorylation of the metabolic master switch AMP-activated protein kinase (AMPK), and decreases glycogen stores in cultured primary murine astrocytes. From these data and our discussion of the literature, mitochondrial sAC emerges as a key regulator of astrocyte bioenergetics. Lastly, we discuss the challenges of investigating the functional and metabolic roles of cytosolic versus mitochondrial sAC in astrocytes employing the currently available pharmacological tool compounds.

Acyclic nucleoside phosphonates with 2-aminothiazole base as inhibitors of bacterial and mammalian adenylate cyclases.

A series of novel acyclic nucleoside phosphonates (ANPs) was synthesized as potential adenylate cyclase inhibitors, where the adenine nucleobase of adefovir (PMEA) was replaced with a 5-substituted 2-aminothiazole moiety. The design was based on the structure of MB05032, a potent and selective inhibitor of fructose 1,6-bisphosphatase and a good mimic of adenosine monophosphate (AMP). From the series of eighteen novel ANPs, which were prepared as phosphoroamidate prodrugs, fourteen compounds were potent (single digit micromolar or submicromolar) inhibitors of Bordetella pertussis adenylate cyclase toxin (ACT), mostly without observed cytotoxicity in J774A.1 macrophage cells. Selected phosphono diphosphates (nucleoside triphosphate analogues) were potent inhibitors of ACT (IC50 as low as 37 nM) and B. anthracis edema factor (IC50 as low as 235 nM) in enzymatic assays. Furthermore, several ANPs were found to be selective mammalian AC1 inhibitors in HEK293 cell-based assays (although with some associated cytotoxicity) and one compound exhibited selective inhibition of mammalian AC2 (only 12% of remaining adenylate cyclase activity) but no observed cytotoxicity. The mammalian AC1 inhibitors may represent potential leads in development of agents for treatment of human inflammatory and neuropathic pain.

Kynurenine Relaxes Arteries of Normotensive Women and Those With Preeclampsia.

[Figure: see text].

Bacillus anthracis edema toxin inhibits hypoxic pulmonary vasoconstriction via edema factor and cAMP-mediated mechanisms in isolated perfused rat lungs.

Bacillus anthracis edema toxin (ET) inhibited lethal toxin-stimulated pulmonary artery pressure (Ppa) and increased lung cAMP levels in our previous study. We therefore examined whether ET inhibits hypoxic pulmonary vasoconstriction (HPV). Following baseline hypoxic measures in isolated perfused lungs from healthy rats, compared with diluent, ET perfusion reduced maximal Ppa increases (mean ± SE percentage of maximal Ppa increase with baseline hypoxia) during 6-min hypoxic periods (FIO2 = 0%) at 120 min (16 ± 6% vs. 51 ± 6%, P = 0.004) and 180 min (11.4% vs. 55 ± 6%, P = 0.01). Protective antigen-mAb (PA-mAb) and adefovir inhibit host cell edema factor uptake and cAMP production, respectively. In lungs perfused with ET following baseline measures, compared with placebo, PA-mAb treatment increased Ppa during hypoxia at 120 and 180 min (56 ± 6% vs. 10 ± 4% and 72 ± 12% vs. 12 ± 3%, respectively, P ≤ 0.01) as did adefovir (84 ± 10% vs. 16.8% and 123 ± 21% vs. 26 ± 11%, respectively, P ≤ 0.01). Compared with diluent, lung perfusion with ET for 180 min reduced the slope of the relationships between Ppa and increasing concentrations of endothelin-1 (ET-1) (21.12 ± 2.96 vs. 3.00 ± 0.76 × 108 cmH2O/M, P < 0.0001) and U46619, a thromboxane A2 analogue (7.15 ± 1.01 vs. 3.74 ± 0.31 × 107 cmH2O/M, P = 0.05) added to perfusate. In lungs isolated from rats after 15 h of in vivo infusions with either diluent, ET alone, or ET with PA-mAb, compared with diluent, the maximal Ppa during hypoxia and the slope of the relationship between change in Ppa and ET-1 concentration added to the perfusate were reduced in lungs from animals challenged with ET alone (P ≤ 0.004) but not with ET and PA-mAb together (P ≥ 0.73). Inhibition of HPV by ET could aggravate hypoxia during anthrax pulmonary infection.NEW & NOTEWORTHY The most important findings here are edema toxin's potent adenyl cyclase activity can interfere with hypoxic pulmonary vasoconstriction, an action that could worsen hypoxemia during invasive anthrax infection with lung involvement. These findings, coupled with other studies showing that lethal toxin can disrupt pulmonary vascular integrity, indicate that both toxins can contribute to pulmonary pathophysiology during infection. In combination, these investigations provide a further basis for the use of antitoxin therapies in patients with worsening invasive anthrax disease.

The Soluble Adenylyl Cyclase Inhibitor LRE1 Prevents Hepatic Ischemia/Reperfusion Damage Through Improvement of Mitochondrial Function.

Hepatic ischemia/reperfusion (I/R) injury is a leading cause of organ dysfunction and failure in numerous pathological and surgical settings. At the core of this issue lies mitochondrial dysfunction. Hence, strategies that prime mitochondria towards damage resilience might prove applicable in a clinical setting. A promising approach has been to induce a mitohormetic response, removing less capable organelles, and replacing them with more competent ones, in preparation for an insult. Recently, a soluble form of adenylyl cyclase (sAC) has been shown to exist within mitochondria, the activation of which improved mitochondrial function. Here, we sought to understand if inhibiting mitochondrial sAC would elicit mitohormesis and protect the liver from I/R injury. Wistar male rats were pretreated with LRE1, a specific sAC inhibitor, prior to the induction of hepatic I/R injury, after which mitochondria were collected and their metabolic function was assessed. We find LRE1 to be an effective inducer of a mitohormetic response based on all parameters tested, a phenomenon that appears to require the activity of the NAD+-dependent sirtuin deacylase (SirT3) and the subsequent deacetylation of mitochondrial proteins. We conclude that LRE1 pretreatment leads to a mitohormetic response that protects mitochondrial function during I/R injury.

Regulation of coral calcification by the acid-base sensing enzyme soluble adenylyl cyclase.

Coral calcification is intricately linked to the chemical composition of the fluid in the extracellular calcifying medium (ECM), which is situated between the calcifying cells and the skeleton. Here we demonstrate that the acid-base sensing enzyme soluble adenylyl cyclase (sAC) is expressed in calcifying cells of the coral Stylophora pistillata. Furthermore, pharmacological inhibition of sAC in coral microcolonies resulted in acidification of the ECM as estimated by the pH-sensitive ratiometric indicator SNARF, and decreased calcification rates, as estimated by calcein labeling of crystal growth. These results indicate that sAC activity modulates some of the molecular machinery involved in producing the coral skeleton, which could include ion-transporting proteins and vesicular transport. To our knowledge this is the first study to directly demonstrate biological regulation of the alkaline pH of the coral ECM and its correlation with calcification.

Signaling pathways involved in anti-inflammatory effects of Pulsed Electromagnetic Field in microglial cells.

Literature studies suggest important protective effects of low-frequency, low-energy pulsed electromagnetic fields (PEMFs) on inflammatory pathways affecting joint and cerebral diseases. However, it is not clear on which bases they affect neuroprotection and the mechanism responsible is yet unknown. Therefore the aim of this study was to identify the molecular targets of PEMFs anti-neuroinflammatory action. The effects of PEMF exposure in cytokine production by lipopolysaccharide (LPS)-activated N9 microglial cells as well as the pathways involved, including adenylyl cyclase (AC), phospholipase C (PLC), protein kinase C epsilon (PKC-ε) and delta (PKC-δ), p38, ERK1/2, JNK1/2 mitogen activated protein kinases (MAPK), Akt and caspase 1, were investigated. In addition, the ability of PEMFs to modulate ROS generation, cell invasion and phagocytosis, was addressed. PEMFs reduced the LPS-increased production of TNF-α and IL-1ß in N9 cells, through a pathway involving JNK1/2. Furthermore, they decreased the LPS-induced release of IL-6, by a mechanism not dependent on AC, PLC, PKC-ε, PKC-δ, p38, ERK1/2, JNK1/2, Akt and caspase 1. Importantly, a significant effect of PEMFs in the reduction of crucial cell functions specific of microglia like ROS generation, cell invasion and phagocytosis was found. PEMFs inhibit neuroinflammation in N9 cells through a mechanism involving, at least in part, the activation of JNK MAPK signalling pathway and may be relevant to treat a variety of diseases characterized by neuroinflammation.

Genome-Wide Small Interfering RNA Screening Reveals a Role for Cullin3-Really Interesting New Gene Ligase Signaling in Heterologous Sensitization of Adenylyl Cyclase.

Heterologous sensitization of adenylyl cyclase (AC) is revealed as enhanced or exaggerated AC/cAMP signaling that occurs following persistent activation of Gα i/o-coupled receptors. This paradoxical phenomenon was discovered more than 40 years ago and was proposed as a cellular mechanism to explain the adaptive changes that occur following chronic exposure to drugs of abuse. However, the underlying molecular mechanisms of heterologous sensitization of AC remain largely unknown. In the present study, we performed a genome-wide cell-based RNA interference screen as an unbiased approach to identify genes associated with heterologous sensitization of AC. Following a series of validation and confirmation assays, three genes that form an E3 ligase complex, cullin3 (CUL3), neural precursor-cell-expressed and developmentally downregulated 8 (NEDD8), and really interesting new gene (RING)-box protein 1 (RBX1), were identified as specific modulators of heterologous sensitization of AC. Furthermore, based on the downstream actions of these genes, we evaluated the activity of proteasome inhibitors as well as the specific NEDD8-activating enzyme inhibitor, MLN4924 (Pevonedistat), in AC sensitization. We demonstrate that MG-132 and bortezomib treatments could mimic the inhibitory effects observed with gene knockdown, and MLN4924 was potent and efficacious in blocking the development of heterologous sensitization of endogenous and recombinant AC isoforms, including AC1, AC2, AC5, and AC6. Together, by using genetic and pharmacological approaches, we identified, for the first time, cullin3-RING ligases and the protein degradation pathway as essential modulators for heterologous sensitization of AC. SIGNIFICANCE STATEMENT: Through a genome-wide cell-based RNA interference screening, we identified three genes that form an E3 ligase complex, cullin3, neural precursor-cell-expressed and developmentally downregulated 8 (NEDD8), and really interesting new gene-box protein 1, as specific modulators of heterologous sensitization of AC. The effect of cullin3, NEDD8, or really interesting new gene-box protein 1 small interfering RNAs on heterologous sensitization was recapitulated by proteasome inhibitors, MG132 and bortezomib, and the specific NEDD8-activating enzyme inhibitor, MLN4924. These results suggest a novel hypothesis in which protein degradation is involved in the sensitization of AC signaling that occurs following chronic activation of Gαi/o-coupled receptors.

The functional association between the sodium/bicarbonate cotransporter (NBC) and the soluble adenylyl cyclase (sAC) modulates cardiac contractility.

The soluble adenylyl cyclase (sAC) was identified in the heart as another source of cyclic AMP (cAMP). However, its cardiac physiological function is unknown. On the other hand, the cardiac Na+/HCO3- cotransporter (NBC) promotes the cellular co-influx of HCO3- and Na+. Since sAC activity is regulated by HCO3-, our purpose was to investigate the potential functional relationship between NBC and sAC in the cardiomyocyte. Rat ventricular myocytes were loaded with Fura-2, Fluo-3, or BCECF to measure Ca2+ transient (Ca2+i) by epifluorescence, Ca2+ sparks frequency (CaSF) by confocal microscopy, or intracellular pH (pHi) by epifluorescence, respectively. Sarcomere or cell shortening was measured with a video camera as an index of contractility. The NBC blocker S0859 (10 µM), the selective inhibitor of sAC KH7 (1 µM), and the PKA inhibitor H89 (0.1 µM) induced a negative inotropic effect which was associated with a decrease in Ca2+i. Since PKA increases Ca2+ release through sarcoplasmic reticulum RyR channels, CaSF was measured as an index of RyR open probability. The generation of CaSF was prevented by KH7. Finally, we investigated the potential role of sAC activation on NBC activity. NBC-mediated recovery from acidosis was faster in the presence of KH7 or H89, suggesting that the pathway sAC-PKA is negatively regulating NBC function, consistent with a negative feedback modulation of the HCO3- influx that activates sAC. In summary, the results demonstrated that the complex NBC-sAC-PKA plays a relevant role in Ca2+ handling and basal cardiac contractility.

Participation of cAMP/PKA-Mediated Signaling Pathways in Functional Activity of Regeneration-Competent Cells in the Nervous Tissue under Conditions of Ethanol-Induced Neurodegeneration.

We studied the involvement of cAMP/PKA signaling in the realization of the growth potential of neural progenitors and secretion of neurotrophic growth factors by glial elements under conditions of ethanol-induced neurodegeneration in vitro and in vivo. The stimulating role of cAMP and PKA in cell cycle progression of the neural progenitor cells and in production of neurotrophins by the cells in nervous tissue under the optimal conditions to vital activity was demonstrated. Ethanol inverted the role of cAMP/PKA signaling pathways in determination of the proliferation-differentiation status of neural stem cells. Selective blockade of adenylate cyclase or PKA in neural stem cells increased the rate of their division against the background of relative decrease in differentiation rate. In addition, cAMP/PKA signaling does not longer participate in neurotrophin production by glial cells in neurodegeneration. These findings suggest that inhibitors of activity/expression of adenylate cyclase and PKA can be considered as possible drugs with regenerative activity for the treatment of nervous system pathologies provoked by alcohol.

Pathological cardiac hypertrophy: the synergy of adenylyl cyclases inhibition in cardiac and immune cells during chronic catecholamine stress.

Response to stressors in our environment and daily lives is an adaptation conserved through evolution as it is beneficial in enhancing the survival and continuity of humans. Although stressors have evolved, the drastic physiological response they elicit still remains unchanged. The chronic secretion and circulation of catecholamines to produce physical responses when they are not required may result in pathological consequences which affect cardiac function drastically. This review seeks to point out the probable implication of chronic stress in inducing an inflammation disorder in the heart. We discussed the likely synergy of a G protein-independent stimuli signaling via ß2-adrenergic receptors in both cardiomyocytes and immune cells during chronic catecholamine stress. To explain this synergy, we hypothesized the possibility of adenylyl cyclases having a regulatory effect on G protein-coupled receptor kinases. This was based on the negative correlations they exhibit during normal cardiac function and heart failures. As such, the downregulation of adenylyl cyclases in cardiomyocytes and immune cells during chronic catecholamine stress enhances the expressions of G protein-coupled receptor kinases. In addition, we explain the maladaptive roles played by G protein-coupled receptor kinase and extracellular signal-regulated kinase in the synergistic cascade that pathologically remodels the heart. Finally, we highlighted the therapeutic potentials of an adenylyl cyclases stimulator to attenuate pathological cardiac hypertrophy (PCH) and improve cardiac function in patients developing cardiac disorders due to chronic catecholamine stress.

The antioxidant resveratrol acts as a non-selective adenosine receptor agonist.

Resveratrol (RSV) is a natural polyphenolic antioxidant with a proven protective role in several human diseases involving oxidative stress, although the molecular mechanism underlying this effect remains unclear. The present work tried to elucidate the molecular mechanism of RSV's role on signal transduction modulation. Our biochemical analysis, including radioligand binding, real time PCR, western blotting and adenylyl cyclase activity, and computational studies provide insights into the RSV binding pathway, kinetics and the most favored binding pose involving adenosine receptors, mainly A2A subtype. In this study, we show that RSV target adenosine receptors (AdoRs), affecting gene expression, receptor levels, and the downstream adenylyl cyclase (AC)/PKA pathway. Our data demonstrate that RSV activates AdoRs. Moreover, RSV activate A2A receptors by directly binding to the classical orthosteric binding site. Intriguingly, RSV-induced receptor activation can stimulate or inhibit AC activity depending on concentration and exposure time. Such subtle and multifaceted regulation of the AdoRs/AC/PKA pathway might contribute to the protective role of RSV. Our findings suggest that RSV molecular action is mediated, at least in part, by activation of adenosine receptors and create the opportunity to interrogate the therapeutic use of RSV in pathological conditions involving AdoRs, such as Alzheimer.

Functional characterization of AC5 gain-of-function variants: Impact on the molecular basis of ADCY5-related dyskinesia.

Adenylyl cyclases are key points for the integration of stimulatory and inhibitory G protein-coupled receptor (GPCR) signals. Adenylyl cyclase type 5 (AC5) is highly expressed in striatal medium spiny neurons (MSNs), and is known to play an important role in mediating striatal dopaminergic signaling. Dopaminergic signaling from the D1 expressing MSNs of the direct pathway, as well as the D2 expressing MSNs of the indirect pathway both function through the regulation of AC5 activity, controlling the production of the 2nd messenger cAMP, and subsequently the downstream effectors. Here, we used a newly developed cell line that used Crispr-Cas9 to eliminate the predominant adenylyl cyclase isoforms to more accurately characterize a series of AC5 gain-of-function mutations which have been identified in ADCY5-related dyskinesias. Our results demonstrate that these AC5 mutants exhibit enhanced activity to Gαs-mediated stimulation in both cell and membrane-based assays. We further show that the increased cAMP response at the membrane effectively translates into increased downstream gene transcription in a neuronal model. Subsequent analysis of inhibitory pathways show that the AC5 mutants exhibit significantly reduced inhibition following D2 dopamine receptor activation. Finally, we demonstrate that an adenylyl cyclase "P-site" inhibitor, SQ22536 may represent an effective future therapeutic mechanism by preferentially inhibiting the overactive AC5 gain-of-function mutants.

Mirabegron induces relaxant effects via cAMP signaling-dependent and -independent pathways in detrusor smooth muscle.

OBJECTIVE: We previously found that mirabegron exerts a relaxant effect in the presence of the ß3 -adrenoceptor antagonist SR58894A during carbachol-induced contraction in human and pig detrusor. The aim of this study was to explore the possible mechanism underlying the relaxant effects of mirabegron using detrusor smooth muscle. METHODS: Human tissue was obtained from urinary bladders of patients undergoing radical cystectomy at Kyushu University and Harasanshin Hospital. Pig tissue was obtained from an abattoir. Tension force (organ bath experiments) was measured in intact or permeabilised (α-toxin or ß-escin) detrusor smooth muscle strips. The contribution of cAMP-dependent signaling and the inhibition of Ca2+ sensitization to the relaxant effects of mirabegron were characterized using 1 µM SR58894A, 100 µM SQ22536 (an adenylyl cyclase inhibitor), 10 µM H-89 (a protein kinase [PK] A inhibitor), 10 µM Y-27632 (a selective Rho kinase inhibitor), and 10 µM GF-109203X (a selective PKC inhibitor). RESULTS: 30 µM Mirabegron impaired carbachol (0.03-1 µM)-induced contraction in human detrusor smooth muscle. SR58894A only partially attenuated the relaxant effects of mirabegron in human and pig detrusor strips precontracted with 1 µM carbachol. In α-toxin-permeabilized detrusor strips, tension force at 1 µM [Ca2+ ]i was decreased by mirabegron in a concentration-dependent manner. The relaxant effect of mirabegron was only slightly attenuated by H-89 and not significantly affected by SQ22536. Y-27632 potentiated the relaxation response to mirabegron, but attenuated responses to cAMP; GF-109203X had little effect. Mirabegron but not cAMP had a notable relaxant effect in the pig detrusor smooth muscle permeabilized with ß-escin. CONCLUSIONS: Mirabegron-induced relaxation of pig and human detrusor smooth muscle occurs via both a ß3 -adrenoceptor/cAMP-dependent and -independent pathway.

Antinociceptive effects of the adenylyl cyclase inhibitor ST034307 on tooth-movement-induced nociception in rats.

OBJECTIVE: This study aimed to investigate the antinociceptive effects of the selective adenylyl cyclase type 1 (AC1) inhibitor ST034307 on tooth movement nociception through orofacial nociceptive behavior tests and molecular examination. METHODS: We placed fixed nickel-titanium alloy closed-coil springs around the incisors of male Sprague-Dawley rats to induce tooth movement. We subsequently administered ST034307 (3 mg/kg), for 2 days, intraperitoneally, and then subjected the rats to a battery of behavioral tests (n = 10/group) to assess orofacial nociception. The changes in the expression of key molecules in the anterior cingulate cortex were measured by ELISA (n = 8/group) and Western blotting (n = 8/group). RESULTS: Tooth movement increased face-grooming activities and rat grimace scale scores. Tooth movement was also associated with enhanced cyclic adenosine monophosphate (cAMP) generation as well as protein kinase A (PKA) activation. Moreover, the phosphorylation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and expression of N-methyl-d-aspartate (NMDA) receptors in the anterior cingulate cortex increased during tooth movement. ST034307 significantly decreased mouth wiping and rat grimace scale scores, accompanied by reductions in cAMP generation, PKA activation, AMPA receptor phosphorylation, and NMDA receptor expression in the anterior cingulate cortex. CONCLUSIONS: These results suggest that adenylyl cyclase type 1 plays an important role in the development of orthodontic tooth movement nociception. Furthermore, ST034307 can be used as an effective pharmacotherapy for orthodontic nociception.