Stimulation Of cAMP Synthesis Research Articles

Control of intracellular pH and bicarbonate by CO2 diffusion into human sperm

The reaction of CO2 with H2O to form bicarbonate (HCO3−) and H+ controls sperm motility and fertilization via HCO3−-stimulated cAMP synthesis. A complex network of signaling proteins participates in this reaction. Here, we identify key players that regulate intracellular pH (pHi) and HCO3− in human sperm by quantitative mass spectrometry (MS) and kinetic patch-clamp fluorometry. The resting pHi is set by amiloride-sensitive Na+/H+ exchange. The sperm-specific putative Na+/H+ exchanger SLC9C1, unlike its sea urchin homologue, is not gated by voltage or cAMP. Transporters and channels implied in HCO3− transport are not detected, and may be present at copy numbers < 10 molecules/sperm cell. Instead, HCO3− is produced by diffusion of CO2 into cells and readjustment of the CO2/HCO3−/H+ equilibrium. The proton channel Hv1 may serve as a unidirectional valve that blunts the acidification ensuing from HCO3− synthesis. This work provides a new framework for the study of male infertility.

PMON181 RGS4 Is Essential For The Sympathetic Regulation of The Short-chain Fatty Acid Receptor FFAR3 In The Heart

Abstract Background Free fatty acids (FFAs) are essential cell nutrients but also important signaling molecules regulating various cellular processes. Short-chain fatty acids (SCFAs), like propionate and butyrate, activate two different types of G protein-coupled receptors (GPCRs), FFAR2 and FFAR3. FFAR3 (also known as GPR41) couples to Gi/o proteins lowering cyclic 3', 5'-adenosine monophosphate (cAMP) levels and activating mitogen-activated protein kinases (MAPKs). In addition to adipose tissue, FFAR3 is highly expressed in peripheral sympathetic ganglia augmenting sympathetic nervous system (SNS) outflow. Regulator of G protein Signaling (RGS)-4 belongs to the RGS superfamily of proteins and inactivates (terminates) both Gi/o- and Gq-protein signaling. Cardiac RGS4 has been implicated in atrial fibrillation (AFib) prevention via calcium signaling attenuation. Finally, β-adrenergic receptors (ARs), the major mediators of catecholaminergic and SNS actions in cardiac myocytes, stimulate cAMP synthesis and activate protein kinase A (PKA), which is known to activate RGS4 via phosphorylation. Objective We examined whether RGS4 regulates cardiac FFAR3 signaling. Methods We used H9c2 cardiomyocytes transfected to express the human FFAR3. After treatment with catecholaminergic agonists and/or propionic acid plus various pharmacological inhibitors and siRNA to deplete RGS4, we performed signaling assays (western blotting, cAMP accumulation, GTP binding, ELISA) to delineate the precise involvement of RGS4 in the cardiac βAR-FFAR3 signaling crosstalk. Results RGS4 is essential for the dampening of propionate/FFAR3-dependent signaling in H9c2 cardiomyocytes since siRNA-mediated RGS4 depletion significantly enhances the effect of propionic acid on cAMP lowering, Gi/o activation, and p38 MAPK activation. Notably, concomitant isoproterenol or salbutamol treatment blocks propionic acid-induced signaling via the FFAR3 in H9c2 cardiomyocytes, indicating negative regulation of FFAR3 by catecholamines via the β2AR. Mechanistically, this is mediated by RGS4, since neither isoproterenol, nor salbutamol can inhibit propionate signaling upon siRNA-mediated RGS4 depletion. Finally, β2AR inhibition of FFAR3 signaling via RGS4 is dependent on β2AR-activated PKA, because pre-treatment with the PKA inhibitor H89 abrogates the catecholaminergic inhibition of FFAR3 signaling in H9c2 cardiomyocytes, as measured by cAMP lowering, downstream p38 MAPK activation, and interleukin (IL)-6 production. Conclusion Catecholamine-activated RGS4 abrogates SCFA/FFAR3 signaling and function in cardiac myocytes. This may represent part of the mechanism via which RGS4 protects against both AFib pathogenesis and FFAR3-promoted SNS hyperactivity and inflammation in the myocardium. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Sphingosine Increases ATP Release From Red Blood Cells

Background: RBC plays a pivotal role in oxygen delivery, improving distribution where it needs. When RBC enters a low oxygen area, a mechanism mediated by a signaling pathway releases ATP, responsible for vasodilatation. Objective: Clarify the potential role of sphingosine on the release of ATP from RBC. Methods: ATP release increases after sphingosine exposure in RBC under low oxygen conditions. ATP release in deoxygenated RBC shows data like that of control RBC: (1) RBC after band 3 modification by 4,4'- diisothio-cyanato-stilbene- 2,2'-disulphonic acid (DIDS); (2) CO-treated RBC. Unlike phosphofructokinase, adenylate cyclase (AC) activity increases after exposure to sphingosine. Results: We show that cAMP synthesis and ATP release are not failed in sphingosine-treated red blood cells in response to incubation with mastoparan 7, forskolin plus 3-isobutyl-1-methyl xanthine, agents that stimulate cAMP synthesis. Conclusion: Deoxy-hemoglobin, band 3, and AC are involved in the signaling pathway responsible for ATP released after sphingosine exposure.

Sphingosine Increases ATP Release From Red Blood Cells

Background: RBC plays a pivotal role in oxygen delivery, improving distribution where it needs. When RBC enters a low oxygen area, a mechanism mediated by a signaling pathway releases ATP, responsible for vasodilatation. Objective: Clarify the potential role of sphingosine on the release of ATP from RBC. Methods: ATP release increases after sphingosine exposure in RBC under low oxygen conditions. ATP release in deoxygenated RBC shows data like that of control RBC: (1) RBC after band 3 modification by 4,4'- diisothio-cyanato-stilbene- 2,2'-disulphonic acid (DIDS); (2) CO-treated RBC. Unlike phosphofructokinase, adenylate cyclase (AC) activity increases after exposure to sphingosine. Results: We show that cAMP synthesis and ATP release are not failed in sphingosine-treated red blood cells in response to incubation with mastoparan 7, forskolin plus 3-isobutyl-1-methyl xanthine, agents that stimulate cAMP synthesis. Conclusion: Deoxy-hemoglobin, band 3, and AC are involved in the signaling pathway responsible for ATP released after sphingosine exposure.

RGS4 Mediates Catecholaminergic Inhibition of Short‐Chain Fatty Acid Receptor FFAR3 Signaling &amp; Function in Cardiomyocytes

BackgroundFree fatty acids (FFAs) are essential cell nutrients but also important signaling molecules regulating various cellular processes. Short‐chain fatty acids (SCFAs), like propionate and butyrate, activate two different types of G protein‐coupled receptors (GPCRs), FFAR2 and FFAR3. FFAR3 (also known as GPR41) couples to Gi/o proteins lowering cyclic 3’,5’‐adenosine monophosphate (cAMP) levels and activating mitogen‐activated protein kinases (MAPKs). In addition to adipose tissue, FFAR3 is highly expressed in peripheral sympathetic ganglia augmenting sympathetic nervous system (SNS) outflow. Regulator of G protein Signaling (RGS)‐4 belongs to the RGS superfamily of proteins and inactivates (terminates) both Gi/o‐ and Gq‐protein signaling. Cardiac RGS4 has been implicated in atrial fibrillation (AFib) prevention via calcium signaling attenuation. Finally, β‐adrenergic receptors (ARs), the major mediators of catecholaminergic and SNS actions in cardiac myocytes, stimulate cAMP synthesis and activate protein kinase A (PKA), which is known to activate RGS4 via phosphorylation.Study objectiveSince catecholamines/SNS and RGS4 play major roles in AFib pathophysiology and FFAR3 promotes SNS activity, we examined whether RGS4 regulates cardiac FFAR3 signaling.MethodsWe used H9c2 cardiomyocytes transfected to express the human FFAR3. After treatment with catecholaminergic agonists and/or propionic acid plus various pharmacological inhibitors and siRNA to deplete RGS4, we performed signaling assays (western blotting, cAMP accumulation, GTP binding, ELISA) to delineate the precise involvement of RGS4 in the cardiac βAR‐FFAR3 signaling crosstalk.ResultsRGS4 is essential for the dampening of propionate/FFAR3‐dependent signaling in H9c2 cardiomyocytes since siRNA‐mediated RGS4 depletion significantly enhances the effect of propionic acid on cAMP lowering, Gi/o activation, and p38 MAPK activation. Notably, concomitant isoproterenol or salbutamol treatment blocks propionic acid‐induced signaling via the FFAR3 in H9c2 cardiomyocytes, indicating negative regulation of FFAR3 by catecholamines via the β2AR. Mechanistically, this is mediated by RGS4, since neither isoproterenol, nor salbutamol can inhibit propionate signaling upon siRNA‐mediated RGS4 depletion. Finally, this β2AR trans‐inhibition of FFAR3 signaling via RGS4 is dependent on β2AR‐activated PKA, because pre‐treatment with the PKA inhibitor H89 abrogated the catecholaminergic inhibition of FFAR3 signaling (cAMP lowering, downstream p38 MAPK activation, interleukin (IL)‐6 production) in H9c2 cardiomyocytes.Conclusionsβ2AR‐activated RGS4 abrogates SCFA/FFAR3 signaling and function in cardiac myocytes. This may represent part of the mechanism via which RGS4 protects against both AFib pathogenesis and FFAR3‐promoted SNS hyperactivity and inflammation in the myocardium.

Melatonin Signaling Pathways Implicated in Metabolic Processes in Human Granulosa Cells (KGN).

Female reproduction depends on the metabolic status, especially during the period of folliculogenesis. Even though it is believed that melatonin can improve oocyte competence, there is still limited knowledge of how it can modulate metabolic processes during folliculogenesis and which signaling pathways are involved in regulating gene expression. To investigate the effects of melatonin on metabolic signals during the antral stage of follicular development, human granulosa-like tumor cells (KGN) were treated with melatonin or forskolin, and gene expression was analyzed with RNA-seq technology. Following appropriate normalization and the application of a fold change cut-off of 1.5 (FC 1.5, p ≤ 0.05), 1009 and 922 genes were identified as differentially expressed in response to melatonin and forskolin, respectively. Analysis of major upstream regulators suggested that melatonin may activate PKB/mTOR signaling pathways to program the metabolism of KGN cells to support slower growth and differentiation and to prevent follicular atresia. Similarly, PKA activation through stimulation of cAMP synthesis with FSK seemed to exert the same effects as melatonin in reducing follicular growth and regulating differentiation. This study suggests that melatonin may act through PKA and PKB simultaneously in human granulosa cells to prevent follicular atresia and early luteinization at the antral stage.

Activation of cAMP-dependent phosphorylation pathways is independent of ROS production during mouse sperm capacitation.

Mammalian sperm have to undergo capacitation to fertilize the egg. At the molecular level, capacitation involves cAMP synthesis, protein kinase A activation, and downstream increase in tyrosine phosphorylation. In addition, during capacitation, mammalian sperm actively generate reactive oxygen species (ROS). It has been proposed that ROS modulate phosphorylation pathways; however, the crosstalk between these signaling processes is not well-understood. In the present study, we used loss- and gain-of-function approaches to evaluate the interconnection between ROS and phosphorylation. We showed that BSA and HCO3- , but not Ca2+ , in the capacitation media are required for ROS production. The synergic effect of these compounds was neither mediated by HCO3- stimulation of cAMP synthesis nor by BSA-induced cholesterol efflux. The capacitation-induced ROS generation was blocked in the presence of superoxide dismutase (SOD), catalase, and apocynin. However, none of these compounds affected cAMP-dependent or tyrosine phosphorylation. On the other hand, the addition of NADPH to the media induced ROS generation in sperm incubated in the absence of BSA and HCO3- without upregulating cAMP-dependent or tyrosine phosphorylation signaling. Most interestingly, catalase, but not SOD, blocked in vitro fertilization suggesting a role for H2 O2 in this process.

Modulation of Cyclic AMP Levels in Fallopian Tube Cells by Natural and Environmental Estrogens.

Autocrine/paracrine factors generated in response to 17β-estradiol (E2) within the fallopian tube (FT) facilitate fertilization and early embryo development for implantation. Since cyclic AMP (cAMP) plays a key role in reproduction, regulation of its synthesis by E2 may be of biological/pathophysiological relevance. Herein, we investigated whether cAMP production in FT cells (FTCs) is regulated by E2 and environmental estrogens (EE’s; xenoestrogens and phytoestrogens). Under basal conditions, low levels of extracellular cAMP were detectable in bovine FTCs (epithelial cells and fibroblasts; 1:1 ratio). Treatment of FTCs with forskolin (AC; adenylyl cyclase activator), isoproterenol (β-adrenoceptor agonist) and IBMX (phosphodiesterase (PDE) inhibitor) dramatically (>10 fold) increased cAMP; whereas LRE1 (sAC; soluble AC inhibitor) and 2’,5’-dideoxyadenosine (DDA; transmembrane AC (tmAC)) inhibitor decreased cAMP. Comparable changes in basal and stimulated intracellular cAMP were also observed. Ro-20-1724 (PDE-IV inhibitor), but not milrinone (PDE-III inhibitor) nor mmIBMX (PDE-I inhibitor), augmented forskolin-stimulated cAMP levels, suggesting that PDE-IV dominates in FTCs. E2 increased cAMP levels and CREB phosphorylation in FTCs, and these effects were mimicked by EE’s (genistein, 4-hydroxy-2’,4’,6’-trichlorobiphenyl, 4-hydroxy-2’,4’,6’-dichlorobiphenyl). Moreover, the effects of E2 and EE were blocked by the tmAC inhibitor DDA, but not by the ERα/β antagonist ICI182780. Moreover, BAPTA-AM (intracellular-Ca2+ chelator) abrogated the effects of E2, but not genistein, on cAMP suggesting differential involvement of Ca2+. Treatment with non-permeable E2-BSA induced cAMP levels and CREB-phosphorylation; moreover, the stimulatory effects of E2 and EEs on cAMP were blocked by G15, a G protein-coupled estrogen receptor (GPER) antagonist. E2 and IBMX induced cAMP formation was inhibited by LRE1 and DDA suggesting involvement of both tmAC and sAC. Our results provide the first evidence that in FTCs, E2 and EE’s stimulate cAMP synthesis via GPER. Exposure of the FT to EE’s and PDE inhibitors may result in abnormal non-cyclic induction of cAMP levels which may induce deleterious effects on reproduction.

Cyclin-Dependent Kinase 18 Controls Trafficking of Aquaporin-2 and Its Abundance through Ubiquitin Ligase STUB1, Which Functions as an AKAP.

Arginine-vasopressin (AVP) facilitates water reabsorption in renal collecting duct principal cells through regulation of the water channel aquaporin-2 (AQP2). The hormone binds to vasopressin V2 receptors (V2R) on the surface of the cells and stimulates cAMP synthesis. The cAMP activates protein kinase A (PKA), which initiates signaling that causes an accumulation of AQP2 in the plasma membrane of the cells facilitating water reabsorption from primary urine and fine-tuning of body water homeostasis. AVP-mediated PKA activation also causes an increase in the AQP2 protein abundance through a mechanism that involves dephosphorylation of AQP2 at serine 261 and a decrease in its poly-ubiquitination. However, the signaling downstream of PKA that controls the localization and abundance of AQP2 is incompletely understood. We carried out an siRNA screen targeting 719 kinase-related genes, representing the majority of the kinases of the human genome and analyzed the effect of the knockdown on AQP2 by high-content imaging and biochemical approaches. The screening identified 13 hits whose knockdown inhibited the AQP2 accumulation in the plasma membrane. Amongst the candidates was the so far hardly characterized cyclin-dependent kinase 18 (CDK18). Our further analysis revealed a hitherto unrecognized signalosome comprising CDK18, an E3 ubiquitin ligase, STUB1 (CHIP), PKA and AQP2 that controls the localization and abundance of AQP2. CDK18 controls AQP2 through phosphorylation at serine 261 and STUB1-mediated ubiquitination. STUB1 functions as an A-kinase anchoring protein (AKAP) tethering PKA to the protein complex and bridging AQP2 and CDK18. The modulation of the protein complex may lead to novel concepts for the treatment of disorders which are caused or are associated with dysregulated AQP2 and for which a satisfactory treatment is not available, e.g., hyponatremia, liver cirrhosis, diabetes insipidus, ADPKD or heart failure.

Discovery of β-arrestin-biased β2-adrenoceptor agonists from 2-amino-2-phenylethanol derivatives.

β-Arrestins are a small family of proteins important for signal transduction at G protein-coupled receptors (GPCRs). β-Arrestins are involved in the desensitization of GPCRs. Recently, biased ligands possessing different efficacies in activating the G protein- versus the β-arrestin-dependent signals downstream of a single GPCR have emerged, which can be used to selectively modulate GPCR signal transduction in such a way that desirable signals are enhanced to produce therapeutic effects while undesirable signals of the same GPCR are suppressed to avoid side effects. In the present study, we evaluated agonist bias for compounds developed along a drug discovery project of β2-adrenoceptor agonists. About 150 compounds, including derivatives of fenoterol, 2-amino-1-phenylethanol and 2-amino-2-phenylethanol, were obtained or synthesized, and initially screened for their β-adrenoceptor-mediated activities in the guinea pig tracheal smooth muscle relaxation assay or thecardiomyocyte contractility assay. Nineteen bioactive compounds were further assessed using both the HTRF cAMP assay and the PathHunter β-arrestin assay. Their concentration-response data in stimulating cAMP synthesis and β-arrestin recruitment were applied to the Black-Leff operational model for ligand bias quantitation. As a result, three compounds (L-2, L-4, and L-12) with the core structure of 5-(1-amino-2-hydroxyethyl)-8-hydroxyquinolin-2(1H)-one were identified as a new series of β-arrestin-biased β2-adrenoceptor agonists, whereas salmeterol was found to be Gs-biased. These findings would facilitate the development of novel drugs for the treatment of both heart failure and asthma.

Improving heterologous expression of porcine follicle-stimulating hormone in Pichia pastoris by integrating molecular strategies and culture condition optimization.

Porcine follicle-stimulating hormone (pFSH), comprising α and β subunits, is commonly used to induce superovulation in domestic animals in assisted reproduction technologies; however, the practical application of pFSH is inhibited by the limited efficiency of its production. Recombinant yeast-derived FSH offers a practical alternative; however, the heterologous expression efficiency remains disappointingly low. To improve FSH production in Pichia pastoris, a series of molecular strategies, together with fermentation optimization, were tested in the present study. By comparing clones of the Muts phenotype strain, it was observed that the yield of soluble pFSH increased by approximately 96% in clones of the Mut+ phenotype strain. The protein levels of soluble pFSHβ, which confers biological specificity, increased by approximately 143 and 22% after two kinds of codon optimization strategies, respectively. Moreover, compared with the production of soluble pFSHβ and SUMO-pFSHβ, the production of soluble protein HSA-pFSHβ was significantly improved. Furthermore, the optimum pH and methanol concentration for expressing soluble HSA-pFSH in strain H3-3 were determined as 5.0-6.0 and 1.5-2% in shake-flask, and the yield of soluble HSA-pFSH could reach 40.8mg/l after purification. In vitro bioactivity assays showed that recombinant HSA-pFSH could efficiently stimulate cAMP synthesis in HEK293 cells expressing porcine FSHR. In conclusion, our results demonstrated that the application of phenotypic selection of aox1 mutants, combined with codon optimization, the choice of fusion partners, and fermentation optimization, considerably increased the yield of pFSH in supernatant of P. pastoris and thus provided a valuable reference for the large-scale recombinant expression of pFSH.

Presynaptic GLP‐1 receptors enhance the depolarization‐evoked release of glutamate and GABA in the mouse cortex and hippocampus

Glucagon-like peptide-1 receptors (GLP-1Rs) have been shown to mediate cognitive-enhancing and neuroprotective effects in the central nervous system. However, little is known about their physiological roles on central neurotransmission, especially at the presynaptic level. Using purified synaptosomal preparations and immunofluorescence techniques, here we show for the first time that GLP-1Rs are localized on mouse cortical and hippocampal synaptic boutons, in particular on glutamatergic and GABAergic nerve terminals. Their activation by the selective agonist exendin-4 (1-100 nM) was able to increase the release of either [3 H]d-aspartate or [3 H]GABA. These effects were abolished by 10 nM of the selective GLP1-R antagonist exendin-3 (9-39) and were prevented by the selective adenylyl cyclase inhibitor 2',5'-dideoxyadenosine (10 µM), indicating the involvement of classic GLP-1Rs coupled to Gs protein stimulating cAMP synthesis. Our data demonstrate the existence and activity of presynaptic receptors for GLP-1 that could represent additional mechanisms by which this neurohormone exerts its effects in the CNS. © 2017 BioFactors, 44(2):148-157, 2018.

Prostanoid EP4 agonist L-902,688 activates PPARγ and attenuates pulmonary arterial hypertension.

Prostacyclin agonists that bind the prostacyclin receptor (IP) to stimulate cAMP synthesis are effective vasodilators for the treatment of idiopathic pulmonary arterial hypertension (IPAH), but this signaling may occur through nuclear peroxisome proliferator-activated receptor-γ (PPARγ). There is evidence of scant IP and PPARγ expression but stable prostanoid EP4 receptor (EP4) expression in IPAH patients. Both IP and EP4 functionally couple with stimulatory G protein (Gs), which activates signal transduction. We investigated the effect of an EP4-specific agonist on pulmonary arterial remodeling and its regulatory mechanisms in pulmonary arterial smooth muscle cells (PASMCs). Immunoblotting evealed IP, EP4, and PPARγ expression in human pulmonary arterial hypertension (PAH) and monocrotaline (MCT)-induced PAH rat lung tissue. Isolated PASMCs from MCT-induced PAH rats (MCT-PASMCs) were treated with L-902,688, a selective EP4 agonist, to investigate the anti-vascular remodeling effect. Scant expression of IP and PPARγ but stable expression of EP4 was observed in IPAH patient lung tissues and MCT-PASMCs. L-902,688 inhibited IP-insufficient MCT-PASMC proliferation and migration by activating PPARγ in a time- and dose-dependent manner, but these effects were reversed by AH-23848 (an EP4 antagonist) and H-89 [a protein kinase A (PKA) inhibitor], highlighting the crucial role of PPARγ in the activity of this EP4 agonist. L-902,688 attenuated pulmonary arterial remodeling in hypoxic PAH mice and MCT-induced PAH rats; therefore, we conclude that the selective EP4 agonist L-902,688 reverses vascular remodeling by activating PPARγ. This study identified a novel EP4-PKA-PPARγ pathway, and we propose EP4 as a potential therapeutic target for PAH.

Dopamine Receptor DAMB Signals via Gq to Mediate Forgetting in Drosophila

Prior studies have shown that aversive olfactory memory is acquired by dopamine acting on a specific receptor, dDA1, expressed by mushroom body neurons. Active forgetting is mediated by dopamine acting on another receptor, Damb, expressed by the same neurons. Surprisingly, prior studies have shown that both receptors stimulate cyclic AMP (cAMP) accumulation, presenting an enigma of how mushroom body neurons distinguish between acquisition and forgetting signals. Here, we surveyed the spectrum of G protein coupling of dDA1 and Damb, and we confirmed that both receptors can couple to Gs to stimulate cAMP synthesis. However, the Damb receptor uniquely activates Gq to mobilize Ca2+ signaling with greater efficiency and dopamine sensitivity. The knockdown of Gαq with RNAi in the mushroom bodies inhibits forgetting but has no effect on acquisition. Our findings identify a Damb/Gq-signaling pathway that stimulates forgetting and resolves the opposing effects of dopamine on acquisition and forgetting.

Adenosine receptors regulate gap junction coupling of the human cerebral microvascular endothelial cells hCMEC/D3 by Ca2+ influx through cyclic nucleotide-gated channels.

Gap junction channels are essential for the formation and regulation of physiological units in tissues by allowing the lateral cell-to-cell diffusion of ions, metabolites and second messengers. Stimulation of the adenosine receptor subtype A2B increases the gap junction coupling in the human blood-brain barrier endothelial cell line hCMEC/D3. Although the increased gap junction coupling is cAMP-dependent, neither the protein kinase A nor the exchange protein directly activated by cAMP were involved in this increase. We found that cAMP activates cyclic nucleotide-gated (CNG) channels and thereby induces a Ca2+ influx, which leads to the increase in gap junction coupling. The report identifies CNG channels as a possible physiological link between adenosine receptors and the regulation of gap junction channels in endothelial cells of the blood-brain barrier. The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the gap junction coupling in endothelial cells of the blood-brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A2A and A2B adenosine receptor subtype agonist 2-phenylaminoadenosine (2-PAA) on the gap junction coupling. We found that 2-PAA stimulated cAMP synthesis and enhanced gap junction coupling in a concentration-dependent manner. This enhancement was accompanied by an increase in gap junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2-PAA-related enhancement of gap junction coupling. In contrast, the cyclic nucleotide-gated (CNG) channel inhibitor l-cis-diltiazem, as well as the chelation of intracellular Ca2+ with BAPTA, or the absence of external Ca2+ , suppressed the 2-PAA-related enhancement of gap junction coupling. Moreover, we observed a 2-PAA-dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca2+ influx by opening CNG channels in a cAMP-dependent manner. Ca2+ in turn induces the formation of new gap junction plaques and a consecutive sustained enhancement of gap junction coupling. The report identifies CNG channels as a physiological link that integrates gap junction coupling into the adenosine receptor-dependent signalling of endothelial cells of the blood-brain barrier.

Cell membrane disruption stimulates cAMP and Ca2+ signaling to potentiate cell membrane resealing in neighboring cells.

ABSTRACTDisruption of cellular plasma membranes is a common event in many animal tissues, and the membranes are usually rapidly resealed. Moreover, repeated membrane disruptions within a single cell reseal faster than the initial wound in a protein kinase A (PKA)- and protein kinase C (PKC)-dependent manner. In addition to wounded cells, recent studies have demonstrated that wounding of Madin-Darby canine kidney (MDCK) cells potentiates membrane resealing in neighboring cells in the short-term by purinergic signaling, and in the long-term by nitric oxide/protein kinase G signaling. In the present study, real-time imaging showed that cell membrane disruption stimulated cAMP synthesis and Ca2+ mobilization from intracellular stores by purinergic signaling in neighboring MDCK cells. Furthermore, inhibition of PKA and PKC suppressed the ATP-mediated short-term potentiation of membrane resealing in neighboring cells. These results suggest that cell membrane disruption stimulates PKA and PKC via purinergic signaling to potentiate cell membrane resealing in neighboring MDCK cells.

Mirabegron relaxes urethral smooth muscle by a dual mechanism involving β3 -adrenoceptor activation and α1 -adrenoceptor blockade.

This article is commented on by Michel, M. C., pp. 429-430 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.13379. Mirabegron is the first β3 -adrenoceptor agonist approved for treatment of overactive bladder syndrome. This study aimed to investigate the effects of β3 -adrenoceptor agonist mirabegron in mouse urethra. The possibility that mirabegron also exerts α1 -adrenoceptor antagonism was also tested in rat smooth muscle preparations presenting α1A - (vas deferens and prostate), α1D - (aorta) and α1B -adrenoceptors (spleen). Functional assays were carried out in mouse and rat isolated tissues. Competition assays for the specific binding of [(3) H]prazosin to membrane preparations of HEK-293 cells expressing each of the human α1 -adrenoceptors, as well as β-adrenoceptor mRNA expression and cyclic AMP measurements in mouse urethra, were performed. Mirabegron produced concentration-dependent urethral relaxations that were shifted to the right by the selective β3 -adrenoceptor antagonist L-748,337 but unaffected by β1 - and β2 -adrenoceptor antagonists (atenolol and ICI-118,551 respectively). Mirabegron-induced relaxations were enhanced by the PDE4 inhibitor rolipram, and the agonist stimulated cAMP synthesis. Mirabegron also produced rightward shifts in urethral contractions induced by the α1 -adrenoceptor agonist phenylephrine. Schild regression analysis revealed that mirabegron behaves as a competitive antagonist of α1 -adrenoceptors in urethra, vas deferens and prostate (α1A -adrenoceptor, pA2 ≅5.6) and aorta (α1D -adrenoceptor, pA2 ≅5.4) but not in spleen (α1B -adrenoceptor). The affinities estimated for mirabegron in functional assays were consistent with those estimated in radioligand binding with human recombinant α1A - and α1D -adrenoceptors (pKi ≅6.0). The effects of mirabegron in urethral smooth muscle are the result of β3 -adrenoceptor agonism together with α1A and α1D -adrenoceptor antagonism.

Regulation of Coupled β-Cell cAMP Dynamics

The islets of Langerhans control glucose homeostasis through secreting insulin. Glucose stimulates insulin secretion via metabolic and electrical events that increase free-calcium activity [Ca2+] to trigger insulin-granule exocytosis. cAMP-regulated pathways also enhancing insulin release. We previously examined how gap junctions coordinate the electrical, [Ca2+] and insulin secretory response. Recent observations have shown that the cAMP is also coordinated, but the mechanisms by which this occurs are poorly understood. To examine this, we measured [Ca2+] and cAMP dynamics in clusters of MIN6 β-cell line and compared results with a multicellular mathematical model of glucose-regulated [Ca2+] and its interaction with cAMP. We find that [Ca2+] and cAMP oscillations are both synchronized across MIN6 clusters. Cell pairs highly synchronized in [Ca2+] were also highly synchronized in cAMP. Cells where cAMP and [Ca2+] oscillations were robust and tightly linked correlated with highly synchronized [Ca2+] and cAMP oscillations. Similar results were observed in the mathematical model that solely contained electrical coupling, supporting that synchronization [Ca2+] is sufficient to promote synchronized cAMP. Optogenetic control of cAMP similarly did not generate coupled cAMP dynamics, unlike optogenetic control of [Ca2+]. Upon Exendin4 stimulated cAMP synthesis, [Ca2+] synchronization increased, which correlated with an increased link between [Ca2+] and cAMP oscillations. This improved link was sufficient to improve [Ca2+] synchronization in the model. An increase in gap junction conductance was not required, and we did not measure a significant increase under the Exendin4 levels used. Therefore cAMP is likely coordinated across the islet indirectly through its interaction with directly coordinated [Ca2+]. In turn cAMP can enhance the coordination of [Ca2+] via changing the [Ca2+] oscillation shape. This yields greater understanding how the signaling underlying insulin release is simultaneously mobilized across the islet and further supports the importance for electrical coupling in islet function.

Inhibition of angiotensin-converting enzyme by aqueous extract of tomato

To investigate the presence of anti-angiotensin converting enzyme (ACE) factors in aqueous extract of tomato. The bio-guided fractionation of the aqueous extract of tomato produced a sugar-free, heat-stable fraction with molecular mass <1,000 Da from tomatoes. The sugar-free tomato extract (TE) was tested for its anti-ACE activity using human plasma and rabbit lung pure ACE. In addition, its effect on human platelet aggregation induced by ADP, collagen or arachidonic acid was determined. The mechanism of platelet inhibitory action of TE was investigated by measuring platelet factor 4 (PF4) release and cAMP synthesis by platelets. Typically, 100 g tomatoes produced 72.2 ± 4.7 mg of TE. This extract inhibited both platelet aggregation and plasma ACE activity in a dose-dependent manner. It inhibited platelet aggregation in response to ADP, collagen or arachidonic acid, and inhibitory action was mediated in part by reducing platelet PF4 release and by stimulating cAMP synthesis. The IC50 value of TE for ADP-induced platelet aggregation was 0.4 ± 0.02 mg/ml, whereas the IC50 value for ACE enzyme inhibition was 1.40 ± 0.04 mg/ml. Both the TE and commercially available sugar-free TE, Fruitflow(®)-2 had similar amount of catechin, and also had equal inhibitory potencies against platelet aggregation and plasma ACE activity. Together these data indicate that aqueous extract of tomatoes contain anti-ACE factors in addition to previously described anti-platelet factors.

Activation of orexin/hypocretin type 1 receptors stimulates cAMP synthesis in primary cultures of rat astrocytes

The effects of orexins, which are also named hypocretins, on cAMP formation were examined in primary cultures of rat astrocytes. Orexin A, an agonist of OX1 and OX2 receptors, stimulated cAMP production with an EC50 value of 0.68µM and potentiated the forskolin-induced increase in the nucleotide synthesis. [Ala11-D-Leu15]orexin B, an agonist of OX2 receptors, was inactive. The effects of orexin A were antagonized by SB 408124, a selective blocker of OX1 receptors, but were not affected by TCS OX2 29, a selective antagonist of OX2 receptors. We hypothesized that the activation of OX1 receptors stimulated cAMP synthesis in primary rat astrocyte cultures.