Intracellular cGMP Accumulation Research Articles

DNA methylation inhibitor regulates guanylyl cyclase/natriuretic peptide receptor-A gene expression via 5'-untranslated region in mesangial and smooth muscle cells

Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) is activated by cardiac hormones atrial and brain natriuretic peptides (ANP, BNP) and produces intracellular second messenger cGMP. The physiological actions of ANP/NPRA/cGMP signaling cascade largely include diuresis, natriuresis, vasorelaxation, and organ protection, which maintains blood pressure and opposed renal, cardiac, and vascular injury and remodeling. However, the mechanisms that contribute to the activation of Npr1 (coding for GC-A/NPRA) expression is not well understood. The present study was aimed at understanding the epigenetic mechanisms of DNA methyltransferase (DNMT) inhibitor, 5-Azacytidine (5-Aza)-mediated Npr1 gene transcription. The studies were carried out in mouse mesangial cells (MMCs) and rat thoracic aortic smooth muscle (RTASM) cells, cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and ITS (insulin, transferrin, and sodium selenite) and treated with 5-Aza for 24 h. Western blot analysis showed that 5-Aza enhanced NPRA protein levels by 5-fold and 7-fold in a dose-dependent manner in MMCs and RTASM cells, respectively, and greatly stimulated intracellular accumulation of cGMP in ANP-treated cells. Treatment with 5-Aza attenuated DNMT activity and repressed DNMT1 protein levels in cells. The MethPrimer search result showed the presence of three cytosine-phosphate-guanine (CpG) islands in the Npr1 full length promoter and 5'-untranslated region (UTR), namely island 1 (-886 to -752, 134 bp), island 2 (-310 to -158, 152 bp), and island 3 (-154 to +310, 464 bp) from the transcription start site. Addition of 5'-UTR encompassing +56 to +359 bp region to the Npr1 promoter constructs, (-1982/+55) and proximal promoter (-356/+55), was inhibitory to Npr1 transcriptional activity, independent of the cell type. Furthermore, treatment with 5-Aza distinctly induced Npr1 promoter activity of the construct -356/5'-UTR by 10-fold in MMCs suggesting that the repressive effect of DNA methylation on Npr1 gene transcription might be via 5'-UTR. The results demonstrate that 5-Aza acts as a potent inducer of Npr1 expression and signaling and provides new insights in the epigenetic control of Npr1, an important player in the control of hypertension and cardiorenal axis. Supported by the NIH grants (HL062147 and DK133833). This is the full abstract presented at the American Physiology Summit 2023 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.

Ligand-Dependent Downregulation of Guanylyl Cyclase/Natriuretic Peptide Receptor-A: Role of miR-128 and miR-195.

Cardiac hormones act on the regulation of blood pressure (BP) and cardiovascular homeostasis. These hormones include atrial and brain natriuretic peptides (ANP, BNP) and activate natriuretic peptide receptor-A (NPRA), which enhance natriuresis, diuresis, and vasorelaxation. In this study, we established the ANP-dependent homologous downregulation of NPRA using human embryonic kidney-293 (HEK-293) cells expressing recombinant receptor and MA-10 cells harboring native endogenous NPRA. The prolonged pretreatment of cells with ANP caused a time- and dose-dependent decrease in 125I-ANP binding, Guanylyl cyclase (GC) activity of receptor, and intracellular accumulation of cGMP leading to downregulation of NPRA. Treatment with ANP (100 nM) for 12 h led to an 80% decrease in 125I-ANP binding to its receptor, and BNP decreased it by 62%. Neither 100 nM c-ANF (truncated ANF) nor C-type natriuretic peptide (CNP) had any effect. ANP (100 nM) treatment also decreased GC activity by 68% and intracellular accumulation cGMP levels by 45%, while the NPRA antagonist A71915 (1 µM) almost completely blocked ANP-dependent downregulation of NPRA. Treatment with the protein kinase G (PKG) stimulator 8-(4-chlorophenylthio)-cGMP (CPT-cGMP) (1 µM) caused a significant increase in 125I-ANP binding, whereas the PKG inhibitor KT 5823 (1 µM) potentiated the effect of ANP on the downregulation of NPRA. The transfection of miR-128 significantly reduced NPRA protein levels by threefold compared to control cells. These results suggest that ligand-dependent mechanisms play important roles in the downregulation of NPRA in target cells.

Phosphodiesterase 5 (PDE5) restricts intracellular cGMP accumulation during enterotoxigenic Escherichia coli infection

ABSTRACT Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) has a continuing impact on residents and travelers in underdeveloped countries. Both heat-labile (LT) and heat-stable (ST) enterotoxins contribute to pathophysiology via induction of cyclic nucleotide synthesis, and previous investigations focused on intracellular signal transduction rather than possible intercellular second messenger signaling. We modeled ETEC infection in human jejunal enteroid/organoid monolayers (HEM) and evaluated cyclic nucleotide pools, finding that intracellular cAMP was significantly increased but also underwent apical export, whereas cGMP was minimally retained intracellularly and predominantly effluxed into the basolateral space. LT and virulence factors including EatA, EtpA, and CfaE promoted ST release and enhanced ST-stimulated cGMP production. Intracellular cGMP was regulated by MK-571-sensitive export in addition to degradation by phosphodiesterase 5. HEMs had limited ST-induced intracellular cGMP accumulation compared to T84 or Caco-2 models. Cyclic nucleotide export/degradation demonstrates additional complexity in the mechanism of ETEC infection and may redirect understanding of diarrheal onset.

Activation of Large Conductance, Calcium-Activated Potassium Channels by Nitric Oxide Mediates Apelin-Induced Relaxation of Isolated Rat Coronary Arteries.

Apelin increases coronary blood flow, cardiac contractility, and cardiac output. Based on these favorable hemodynamic effects, apelin and apelin-like analogs are being developed for treating heart failure and related disorders; however, the molecular mechanisms underlying apelin-induced coronary vasodilation are unknown. This study aimed to elucidate the signaling pathways by which apelin causes smooth muscle relaxation in coronary arteries. Receptors for apelin (APJ receptors) were expressed in coronary arteries, as determined by Western blot and polymerase chain reaction analyses. Immunofluorescence imaging studies identified APJ receptors on endothelial and smooth muscle cells. In isolated endothelial cells, apelin caused an increase in 4,5-diaminofluorescein fluorescence that was abolished by nitro-l-arginine (NLA) and F13A (H-Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Ala-OH), an APJ receptor antagonist, consistent with increased nitric oxide (NO) production. In arterial rings, apelin caused endothelium-dependent relaxations that were abolished by NLA, F13A, and iberiotoxin. Neither oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) nor DT-2, a protein kinase G inhibitor, had any effect on apelin-induced relaxations, and apelin itself had no effect on intracellular cGMP accumulation in coronary arteries. Patch-clamp studies in isolated smooth muscle cells demonstrated that the NO donors, diethyl amine NONOate and sodium nitroprusside, caused increases in large conductance, calcium-activated potassium channel (BKCa) currents, which were inhibited by iberiotoxin but not ODQ. Thus, apelin causes endothelium-dependent relaxation of coronary arteries by stimulating endothelial APJ receptors and releasing NO, which acts in a cGMP-independent manner and increases BKCa activity in the underlying smooth muscle cells. These results provide a mechanistic basis for apelin-induced coronary vasodilation and may provide guidance for the future development of novel apelin-like therapeutic agents.

Apelin and Acetylcholine Activate Distinct Nitric Oxide Signaling Pathways to Cause Endothelium‐Dependent Relaxation of Isolated Coronary Arteries

The peptide, apelin, is expressed in fat cells, endothelial cells, and CNS neurons. Increasing evidence (e.g. inotropic and vasomotor effects) supports a role for apelin in the regulation of the cardiovascular system. Apelin is reported to cause vasodilation in peripheral arteries but the underlying molecular mechanisms are not well understood. In the present study, we investigated the role of nitric oxide (NO) in apelin‐induced relaxation of isolated rat coronary arteries, and compared it with the classic endothelium‐dependent vasodilator, acetylcholine. Receptors for apelin (APJ receptors) were expressed in coronary arteries, as determined by Western blot and RT‐PCR analysis (n=3). Immunofluorescence staining and confocal microscopy demonstrated the presence of APJ receptors on endothelial and smooth muscle cells in the coronary arterial wall. In freshly isolated coronary endothelial cells, apelin (10−6 M) caused an increase in DAF‐2 fluorescence that was abolished by nitro‐l‐arginine (3 × 10−5 M; NLA) and F13A (10−7 M), an APJ receptor antagonist, consistent with an increase in NO production. In coronary arterial rings contracted with 5‐HT (10−7 M), apelin caused endothelium‐dependent relaxations (pD2=6.90 ± 0.1; Emax=45 ± 6%; n=9) that were abolished in the presence of NLA, F13A, or iberiotoxin (10−7 M; IBTx), as well as in rings contracted with a depolarizing concentration of K+ (40 mM). Neither ODQ (10−5 M) nor DT‐2 (10−6 M), a protein kinase G (PKG) inhibitor, had any effect on apelin‐induced relaxations, and apelin (10−6 M) itself had no effect on intracellular cGMP accumulation in coronary arteries (0.09 ± 0.03 vs 0.05 ± 0.01 pmol/mg; n=4; p>0.05). By contrast, endothelium‐dependent relaxations to acetylcholine (pD2=6.89 ± 0.1; Emax=97 ± 2%; n=8) were inhibited by NLA, ODQ, and DT‐2. Acetylcholine (10−6 M) increased DAF‐2 fluorescence in isolated endothelial cells, and increased cGMP accumulation (0.08 ± 0.04 vs 0.26 ± 0.02 pmol/mg; n=4; p<0.05) in coronary arteries in an NLA‐ and ODQ‐sensitive manner. Patch clamp studies in freshly isolated coronary smooth muscle cells demonstrated that the NO donor, DEA NONOate (10−6 M) caused an increase in large conductance, calcium‐activated K (BKCa) current (peak current density (+80mV) = 53.02 ± 10.4 vs. 90.42 ± 8.6 pA/pF without and with DEA NONOate, respectively; n=6; p<0.05), that was inhibited by IBTx (peak current density = 33.8 ± 5.3 pA/pF; n=6; p<0.05 vs DEA NONOate alone) but not by ODQ (peak current density = 90.2 ± 7.9 pA/pF; n=6; p>0.05 vs DEA NONOate alone). Taken together, the data demonstrate that, in coronary arteries, endothelium‐derived NO causes arterial relaxation via two distinct signaling pathways: 1) which is activated by apelin, is sensitive to changes in membrane potential and requires activation of BKCa channels; this pathway is independent of cGMP formation and may be due to direct activation of BKCa channels by NO; and 2) which is activated by acetylcholine, is cGMP‐dependent and is mediated by activation of PKG. Differential regulation of distinct NO‐signaling pathways may provide opportunities for developing novel therapeutic strategies for the treatment of the coronary arterial dysfunction that often occurs in cardiovascular disease.Support or Funding InformationSupported by a grant from the NIH National Heart, Lung and Blood Institute (HL124338)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

How Excessive cGMP Impacts Metabolic Proteins in Retinas at the Onset of Degeneration.

Aryl-hydrocarbon receptor interacting protein-like 1 (AIPL1) is essential to stabilize cGMP phosphodiesterase 6 (PDE6) in rod photoreceptors. Mutation of AIPL1 leads to loss of PDE6, accumulation of intracellular cGMP, and rapid degeneration of rods. To understand the metabolic basis for the photoreceptor degeneration caused by excessive cGMP, we performed proteomics and phosphoproteomics analyses on retinas from AIPL1-/- mice at the onset of rod cell death. AIPL1-/- retinas have about 18 times less than normal PDE6a and no detectable PDE6b. We identified twelve other proteins and thirty-nine phosphorylated proteins related to cell metabolism that are significantly altered preceding the massive degeneration of rods. They include transporters, kinases, phosphatases, transferases, and proteins involved in mitochondrial bioenergetics and metabolism of glucose, lipids, amino acids, nucleotides, and RNA. In AIPLI-/- retinas mTOR and proteins involved in mitochondrial energy production and lipid synthesis are more dephosphorylated, but glycolysis proteins and proteins involved in leucine catabolism are more phosphorylated than in normal retinas. Our findings indicate that elevating cGMP rewires cellular metabolism prior to photoreceptor degeneration and that targeting metabolism may be a productive strategy to prevent or slow retinal degeneration.

Abstract P195: Transforming Growth Factor beta 1-Mediated Repression of Guanylyl Cyclase/Natriuretic Peptide Receptor-A Gene Expression and Function Involving TGF-beta 1 and Delta EF1 in Vascular Smooth Muscle Cells

The binding of atrial and brain natriuretic peptides (ANP and BNP) to guanylyl cyclase-A/natriuretic peptide receptor-A (GC-A/NPR-A) produces second messenger cGMP, which lowers blood pressure and prevents cardiovascular events. The objective of the present study was to examine the repressive effect of transforming growth factor (TGF-β1) in the regulation of Npr1 (coding for GC-A/NPRA) gene expression and function. The rat thoracic aortic vascular smooth muscle cells (RTASMC) were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and treated with TGF-β1. The luciferase assay results showed that TGF-β1 significantly repressed Npr1 promoter activity in a dose- and time-dependent manner by 82% and 85% (2.5 ng/ml, 25.51 ± 2.2 and 24 h, 7.0 ± 0.6 vs. untreated control 70.06 ± 4.6, p < 0.001, respectively). Treatment with TGF-β1 decreased NPRA mRNA and protein levels by 62% (treated, 0.42 ± 0.05 vs. control, 0.9 ± 0.02, p < 0.01) and 55% (treated, 9603 ± 860 vs. untreated, 22211 ± 1449, p < 0.01), respectively. TGF-β1 attenuated ANP-dependent intracellular accumulation of cGMP by 59% (TGF-β1 + ANP-treated, 8.66 ± 0.9 pmol/1x 106 cells vs. ANP-treated cells 23.51 ± 2.2; p < 0.001). Chromatin immunoprecipitation and electrophoretic mobility shift assay showed that TGF-β1 enhanced the recruitment of transcription factor delta EF1 (δEF1) to form a transcriptional repressor complex with their binding sites in Npr1 promoter. Western blot analysis showed significant increase in δEF1 protein expression by 2.4-fold (treated, 907.9 ± 36.5. vs. untreated, 378.5 ± 10.3; p < 0.001) and phosphorylation of mothers against decapentaplegic homolog 2/3 (SMAD2/3) proteins by 2.3-fold (treated, 620.9 ± 10.6 vs. untreated, 269 ± 9.7; p < 0.01) in TGF-β1-treated cells. Collectively, the present results demonstrate that TGF-β1 represses Npr1 gene transcription and expression by interactive actions of δEF1 and phosphorylated SMADs. Together, the present results suggest that TGF-β1 and δEF1 inhibit Npr1 gene expression and function, which may be critical in regulating the blood pressure and cardiovascular homeostasis.

MRP4 Modulation of the Guanylate Cyclase-C/cGMP Pathway: Effects on Linaclotide-Induced Electrolyte Secretion and cGMP Efflux.

MRP4 mediates the efflux of cGMP and cAMP and acts as an important regulator of these secondary messengers, thereby affecting signaling events mediated by cGMP and cAMP. Immunofluorescence staining showed high MRP4 expression localized predominantly in the apical membrane of rat colonic epithelium. In vitro studies were performed using a rat colonic mucosal layer mounted in an Ussing chamber. Linaclotide activation of the guanylate cyclase-C (GC-C)/cGMP pathway induced a concentration-dependent increase in transepithelial ion current [short-circuit current (Isc)] across rat colonic mucosa (EC50: 9.2 nM). Pretreatment of colonic mucosa with the specific MRP4 inhibitor MK571 potentiated linaclotide-induced electrolyte secretion and augmented linaclotide-stimulated intracellular cGMP accumulation. Notably, pretreatment with the phosphodiesterase 5 inhibitor sildenafil increased basal Isc, but had no amplifying effect on linaclotide-induced Isc. MRP4 inhibition selectively affected the activation phase, but not the deactivation phase, of linaclotide. In contrast, incubation with a GC-C/Fc chimera binding to linaclotide abrogated linaclotide-induced Isc, returning to baseline. Furthermore, linaclotide activation of GC-C induced cGMP secretion from the apical and basolateral membranes of colonic epithelium. MRP4 inhibition blocked cGMP efflux from the apical membrane, but not the basolateral membrane. These data reveal a novel, previously unrecognized mechanism that functionally couples GC-C-induced luminal electrolyte transport and cGMP secretion to spatially restricted, compartmentalized regulation by MRP4 at the apical membrane of intestinal epithelium. These findings have important implications for gastrointestinal disorders with symptoms associated with dysregulated fluid homeostasis, such as irritable bowel syndrome with constipation, chronic idiopathic constipation, and secretory diarrhea.

Carboxyamidotriazole: A novel inhibitor of both cAMP-phosphodiesterases and cGMP-phosphodiesterases

Carboxyamidotriazole (CAI) is a non-cytotoxic anti-tumor drug, which also shows considerable anti-inflammatory effects in a variety of animal models of inflammation. The exact target and mechanism of CAI were not clearly understood yet. In the present study, we demonstrate that CAI is a non-selective phosphodiesterase (PDE) inhibitor, which provides comprehensive inhibitions of both adenosine 3′,5′-cyclic monophosphate specific PDE (cAMP-PDE) and guanosine 3′,5′-cyclic monophosphate specific PDE (cGMP-PDE) isolated from rat brain, mouse pulmonary tissue, primary mouse peritoneal macrophages, RAW264.7 cells, Lewis lung carcinoma (LLC) cells and lymphocytic leukemia cells (L1210) with moderate potencies (IC50≈0.5–30μM). The comprehensive elimination of PDE activities in living LLC cells by CAI results in accumulation of intracellular cAMP and cGMP, which can be visualized by fluorescence resonance energy transfer (FRET)-based cyclic nucleotide sensors. The stimulation by 30μM CAI yielded ~1.5-fold greater cGMP responses compared with 10μM sildenafil citrate, whereas the influence of 30μM CAI on cAMP levels was similar as that of 100μM 3-isobutyl-1-methylxanthine (IBMX). The non-selective inhibitory effect of CAI on cAMP-PDE and cGMP-PDE increases the likelihood for CAI to affect the balance between the levels of intracellular cyclic nucleotides cAMP and cGMP, then a variety of cellular signaling pathways that regulate cell functions and even related disease processes. When examining the widely proven anti-tumor and anti-inflammatory activities of CAI, it is important to affirm its comprehensive inhibitory effect on PDEs, which makes it superior to some selective PDE inhibitors in a way.

Guanylyl cyclase/natriuretic peptide receptor-A signaling antagonizes phosphoinositide hydrolysis, Ca(2+) release, and activation of protein kinase C.

Thus far, three related natriuretic peptides (NPs) and three distinct sub-types of cognate NP receptors have been identified and characterized based on the specific ligand binding affinities, guanylyl cyclase activity, and generation of intracellular cGMP. Atrial and brain natriuretic peptides (ANP and BNP) specifically bind and activate guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), and C-type natriuretic peptide (CNP) shows specificity to activate guanylyl cyclase/natriuretic peptide receptor-B (GC-B/NPRB). All three NPs bind to natriuretic peptide receptor-C (NPRC), which is also known as clearance or silent receptor. The NPRA is considered the principal biologically active receptor of NP family; however, the molecular signaling mechanisms of NP receptors are not well understood. The activation of NPRA and NPRB produces the intracellular second messenger cGMP, which serves as the major signaling molecule of all three NPs. The activation of NPRB in response to CNP also produces the intracellular cGMP; however, at lower magnitude than that of NPRA, which is activated by ANP and BNP. In addition to enhanced accumulation of intracellular cGMP in response to all three NPs, the levels of cAMP, Ca2+ and inositol triphosphate (IP3) have also been reported to be altered in different cells and tissue types. Interestingly, ANP has been found to lower the concentrations of cAMP, Ca2+, and IP3; however, NPRC has been proposed to increase the levels of these metabolic signaling molecules. The mechanistic studies of decreased and/or increased levels of cAMP, Ca2+, and IP3 in response to NPs and their receptors have not yet been clearly established. This review focuses on the signaling mechanisms of ANP/NPRA and their biological effects involving an increased level of intracellular accumulation of cGMP and a decreased level of cAMP, Ca2+, and IP3 in different cells and tissue systems.

Abstract 282: Aromatic Residue-based Tetrapeptide Fqqi Motif Mediates Internalization and Trafficking of Guanylyl-cyclase/natriuretic Peptide Receptor-a in Mouse Mesangial Cells

Atrial natriuretic peptide (ANP) is a cardiac hormone that modulates blood pressure and blood volume by activation of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) and subsequent generation of intracellular second messenger cGMP. The aim of the present study was to examine the role of small sequence motifs in the internalization and trafficking pathway of NPRA by immunofluorescence confocal microscopy. A chimeric protein of enhanced green fluorescence protein (eGFP) and NPRA (eGFP-NPRA) was used to study the dynamics of NPRA trafficking in the intact mouse mesangial cells (MMCs). We have identified a FQQI (Phe 790 , Gln 791 , Gln 792 and Ile 793 ) motif in the carboxyl-terminal domain of NPRA sequence and mutated with alanine residues (FQQI/AQQA) using QuickChange II site-directed mutagenesis kit. Cells were transiently transfected with mutated and wild-type constructs and cultured at 37 0 C in an atmosphere of 5% CO 2 and 95% O 2 . In the ANP-treated cells, colocalization of wild-type eGFP-NPRA with pan-Cadherin, a plasma membrane marker, indicated that internalization of receptor accounted for 59.2%±3.4, 70.0%±4.3, 71.8%±4.3, and 77.4%±3.2 at 5, 10, 15, and 30 min, respectively of total fluorescence intensity compared with controls. Colocalization of eGFP-NPRA with early endosome antigen-1 (EEA-1) marker was maximum at 5 min (64.5%±2.3), and gradually decreased at 30 min (24.4%±3.8). However, the mutation of FQQI/AQQA decreased the receptor internalization by 45% ( P< 0.001) and the receptor colocalization in to endosome by 58% ( P <0.0001), respectively, compared with wild type receptor. After ANP treatment, immunofluorescence analysis showed a marked increase in the accumulation of intracellular cGMP (29-fold) concurrent with receptor internalization compared with controls. The results indicate that ligand-receptor complexes continue to traffic and produce cGMP even after receptor sequestration into endosomes. Our, data demonstrate that FQQI motif plays essential role in the internalization of NPRA, which regulates hypertension and cardiovascular homeostasis.

Decreasing phosphodiesterase 5A activity contributes to platelet cGMP accumulation during storage of apheresis‐derived platelet concentrates

Platelet storage lesion (PSL) considerably decreases the quality of platelets (PLTs) in concentrates characterized by a loss of signaling responses to agonists and impaired PLT activation, secretion, and aggregation. To understand the role of inhibitory signaling pathways in the mechanism of PSL, the basal state of the cyclic nucleotide (CN)-dependent signaling systems in stored PLTs was investigated. Whole blood samples (WB) and apheresis-derived PLT concentrates (APCs) were obtained from healthy volunteers. Washed PLTs were prepared from WB on Day 0 and from APCs on Days 0, 2, and 5. The basal phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) and phosphodiesterase 5A (PDE5A) levels were quantified by Western blot. CN and PDE5A activity were measured by enzyme-linked immunoassay kits. Fibrinogen binding and aggregation were measured in PLT-rich plasma of WB or APC samples. Unpaired t test was used for statistical analysis. Basal VASP phosphorylation levels were comparable in WB and APCs on Day 0. VASP phosphorylation increased significantly during storage of APCs, more pronounced at Ser(239) than at Ser(157) . Similarly, intracellular cGMP, but not cAMP, concentration continuously increased in stored PLTs, whereas PDE5A levels and activity significantly decreased accompanied by diminished thrombin receptor activator peptide 6-induced fibrinogen binding and aggregation. Storage of APCs leads to intracellular cGMP accumulation that could be caused by degradation of PDE5A. Enhanced cGMP level supports subsequent cGMP-dependent protein kinase-mediated increase of VASP phosphorylation resulting in reduced fibrinogen binding and aggregation.

Bile Acid Receptor TGR5 Agonism Induces NO Production and Reduces Monocyte Adhesion in Vascular Endothelial Cells

TGR5 is a G-protein-coupled receptor for bile acids. So far, little is known about the function of TGR5 in vascular endothelial cells. In bovine aortic endothelial cells, treatment with a bile acid having a high affinity to TGR5, taurolithocholic acid (TLCA), significantly increased NO production. This effect was abolished by small interfering RNA-mediated depletion of TGR5. TLCA-induced NO production was also observed in human umbilical vein endothelial cells measured via intracellular cGMP accumulation. TLCA increased endothelial NO synthase(ser1177) phosphorylation in human umbilical vein endothelial cells. This response was accompanied by increased Akt(ser473) phosphorylation and intracellular Ca(2+). Inhibition of these signals significantly decreased TLCA-induced NO production. We next examined whether TGR5-mediated NO production affects inflammatory responses of endothelial cells. In human umbilical vein endothelial cells, TLCA significantly reduced tumor necrosis factor-α-induced adhesion of monocytes, vascular cell adhesion molecule-1 expression, and activation of nuclear factor-κB. TLCA also inhibited lipopolysaccharide-induced monocyte adhesion to mesenteric venules in vivo. These inhibitory effects of TLCA were abrogated by NO synthase inhibition. TGR5 agonism induces NO production via Akt activation and intracellular Ca(2+) increase in vascular endothelial cells, and this function inhibits monocyte adhesion in response to inflammatory stimuli.

Melatonin inhibits nitric oxide signaling by increasing PDE5 phosphorylation in coronary arteries

Melatonin inhibits nitric oxide (NO)-induced relaxation of coronary arteries. We tested the hypothesis that melatonin increases the phosphorylation of phosphodiesterase 5 (PDE5), which increases the activity of the enzyme and thereby decreases intracellular cGMP accumulation in response to NO and inhibits NO-induced relaxation. Sodium nitroprusside (SNP) and 8-Br-cGMP caused concentration-dependent relaxation of isolated coronary arteries suspended in organ chambers for isometric tension recording. In the presence of melatonin, the concentration-response curve to SNP, but not 8-Br-cGMP, was shifted to the right. The effect of melatonin on SNP-induced relaxation was abolished in the presence of the PDE5 inhibitors zaprinast and sildenafil. Melatonin markedly inhibited the SNP-induced increase in intracellular cGMP in coronary arteries, an effect that was also abolished by zaprinast. Treatment of coronary arteries with melatonin caused a nearly fourfold increase in the phosphorylation of PDE5, which increased the catalytic activity of the enzyme and thereby increased the degradation of cGMP to inactive 5'-GMP. Melatonin-induced PDE5 phosphorylation was markedly attenuated in the presence of the PKG1 inhibitors DT-2 or Rp-8-Br-PET-cGMPS and in those arteries in which PKG1 expression was first downregulated by 24-h incubation with SNP before exposure to melatonin. The selective MT(2) receptor antagonist 4-phenyl-2-propionamidotetralin completely blocked the stimulatory effect of melatonin on PDE5 phosphorylation as well as the inhibitory effect of melatonin on SNP-induced relaxation and intracellular cGMP. Thus, in coronary arteries, melatonin acts via MT(2) receptors and PKG1 to increase PDE5 phosphorylation, resulting in decreased cGMP accumulation in response to NO and impaired NO-induced vasorelaxation.

A new method for establishing stable cell lines and its use for large-scale production of human guanylyl cyclase-B receptor and of the extracellular domain

Guanylyl cyclase-B receptor (GC-B) is a membrane receptor that induces intracellular accumulation of cGMP when a specific ligand, C-type natriuretic peptide (CNP), binds to the extracellular ligand-binding domain (ECD). Despite of its medical and biological importance, characterization of GC-B is hampered by limited amounts of protein obtainable. To circumvent this problem, a method was developed for rapidly and semi-automatically establishing stable cell lines specialized for large-scale production. This method, utilizing a bicistronic expression vector for co-expressing a green fluorescent protein and FACS-based selection of high-expressing cells, is generally applicable. It worked particularly well with the ECD and yielded highly purified ECD at 1mg/l of culture medium by affinity chromatography using modified CNPs. Measurements of ligand-binding and guanylyl cyclase activities for various natriuretic peptides showed that, as expected, CNP is by far the most potent agonist of GC-B with IC50 of ∼7.5nM. This value is at least an order of magnitude larger than that reported earlier but similar to that established with the guanylyl cyclase-A receptor for its ligand, atrial natriuretic peptide. The methods developed here will be useful, at the least, for characterizing other members of the guanylyl cyclase receptor family.

Cytosolic expression of aldehyde dehydrogenase-2 is essential for nitroglycerin bioactivation by cultured cells

We have recently shown that aldehyde dehydrogenase-2 (ALDH2) is mainly cytosolic in murine vascular tissue. Relaxation of ALDH2 knockout aortas to nitroglycerin (GTN) was restored by cytosolic but not by mitochondrial overexpression of ALDH2. To find out whether GTN bioactivation is determined by the endogenous distribution of ALDH2, we studied two types of cultured cells with strictly different ALDH2 localization for GTN-induced formation of 1,2-glycerol dinitrate (1,2-GDN) and accumulation of intracellular cGMP. Chloral hydrate (1 mM) and daidzin (0.1 mM) were used as ALDH inhibitors. Quantitative immunoblotting revealed that porcine aortic endothelial cells (PAECs) and rat lung fibroblasts (RFL-6 cells) expressed the protein predominantly in mitochondrial and cytosolic fractions, respectively. Interestingly, ALDH2-catalyzed GTN metabolism required addition of dithiothreitol (DTT), indicating that the cultured cells lack an essential reductant that is present in blood vessels. In the presence of 10 μM GTN and 2 mM DTT, the rates of 1,2-GDN formation were 0.5 ± 0.2 and 1.3 ± 0.4 pmol min −1 mg −1 in PAECs and RFL-6 cells, respectively. The maximal cGMP levels observed with 10 μM GTN in the presence of 2 mM DTT and 1 kU SOD/ml were 26 ± 4 and 87 ± 16 pmol/10 6 cell in PAECs and RFL-6 cells, corresponding to about 2- and 5-fold increases of basal levels, respectively. These results show that cytosolic expression of ALDH2 is essential for GTN bioactivation, indicating limited access of GTN to the mitochondrial matrix. Further work is needed to identify the endogenous reductant that was replaced by DTT in the present study.

Pseudomonas aeruginosa Exotoxin Y Is a Promiscuous Cyclase That Increases Endothelial Tau Phosphorylation and Permeability

Exotoxin Y (ExoY) is a type III secretion system effector found in ~ 90% of the Pseudomonas aeruginosa isolates. Although it is known that ExoY causes inter-endothelial gaps and vascular leak, the mechanisms by which this occurs are poorly understood. Using both a bacteria-delivered and a codon-optimized conditionally expressed ExoY, we report that this toxin is a dual soluble adenylyl and guanylyl cyclase that results in intracellular cAMP and cGMP accumulation. The enzymatic activity of ExoY caused phosphorylation of endothelial Tau serine 214, accumulation of insoluble Tau, inter-endothelial cell gap formation, and increased macromolecular permeability. To discern whether the cAMP or cGMP signal was responsible for Tau phosphorylation and barrier disruption, pulmonary microvascular endothelial cells were engineered for the conditional expression of either wild-type guanylyl cyclase, which synthesizes cGMP, or a mutated guanylyl cyclase, which synthesizes cAMP. Sodium nitroprusside stimulation of the cGMP-generating cyclase resulted in transient Tau serine 214 phosphorylation and gap formation, whereas stimulation of the cAMP-generating cyclase induced a robust increase in Tau serine 214 phosphorylation, gap formation, and macromolecular permeability. These results indicate that the cAMP signal is the dominant stimulus for Tau phosphorylation. Hence, ExoY is a promiscuous cyclase and edema factor that uses cAMP and, to some extent, cGMP to induce the hyperphosphorylation and insolubility of endothelial Tau. Because hyperphosphorylated and insoluble Tau are hallmarks in neurodegenerative tauopathies such as Alzheimer disease, acute Pseudomonas infections cause a pathophysiological sequela in endothelium previously recognized only in chronic neurodegenerative diseases.

Abstract 153: Bile Acid Receptor TGR5 Agonism Represents Anti-inflammatory Effects via Stimulating Nitric Oxide Production in Vascular Endothelial Cells

Objective TGR5, a membrane-bound, G-protein-coupled receptor for bile acids, is known to be involved in regulation of energy homeostasis and inflammation. However, little is known about the function of TGR5 in vascular endothelial cells. In the present study, we examined whether TGR5 agonism represents anti-inflammatory effects in vascular endothelial cells focusing on nitric oxide (NO) production. Methods and Results In human umbilical vein endothelial cells (HUVECs), treatment with taurolithocholic acid (TLCA), which has the highest affinity to TGR5 among various bile acids, significantly reduced tumor necrosis factor (TNF)-α-induced vascular cell adhesion molecule (VCAM)-1 protein expression and adhesion of human monocytes, U937. These effects were abrogated by a NO synthase (NOS) inhibitor, N G -Monomethyl-L-arginine (L-NMMA). In bovine aortic endothelial cells (BAECs), treatment with TLCA as well as lithocholic acid, which also has high affinity to TGR5, significantly increased the NO production. In contrast, deoxycholic acid and chenodeoxycholic acid, which possess low affinity to TGR5, did not affect the NO production. Gene depletion of TGR5 by siRNA transfection abolished TLCA-induced NO production in BAECs. TLCA-induced NO production was also observed in HUVECs measured as intracellular cGMP accumulation. We next investigated the signal pathways responsible for the TLCA-induced NO production in endothelial cells. Treatment with TLCA increased endothelial NOS (eNOS) ser1177 phosphorylation in HUVECs. This response was accompanied by increased Akt ser473 phosphorylation and intracellular Ca 2+ ([Ca 2+ ] i ). Treatment with phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, or blockade of calcium channel with La 3+ , significantly decreased TLCA-induced eNOS ser1177 phosphorylation and subsequent NO production. Conclusion These results indicate that TGR5 agonism can mediate anti-inflammatory responses by suppressing VCAM-1 expression and monocytes adhesion to endothelial cells. This function is dependent on NO production via Akt activation and [Ca 2+ ] i increase.

Abstract 570: Breast cancer chemopreventive activity of a novel amide derivative of sulindac involves inhibition of PDE5, activation of PKG, and attenuation of Wnt/β-catenin signaling

Abstract The nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase-2 (COX2) inhibitors demonstrate promising chemopreventive activity against a number of cancer types, including breast cancer. Unfortunately, potentially fatal toxicities associated with COX inhibition have precluded the development of these drugs for such indications. However, some studies suggest that their anticancer activity may be due to COX-independent mechanisms, suggesting the ability to target such mechanisms for the development of potentially safer and more effective chemopreventive agents. Here we describe a novel amide derivative of the NSAID sulindac sulfide (SS), termed sulindac sulfide amide (SSA). SSA lacked the ability to inhibit either COX1 or COX2 but demonstrated as much as a twenty-fold increase in potency to inhibit growth and induce apoptosis of breast tumor cells compared to SS. Moreover, SSA was selective for tumor cells, having no measurable effect on apoptosis of nontumorigenic mammary epithelial cells. As we have shown previously with SS, SSA inhibited the cGMP specific phosphodiesterase 5 (PDE5) enzyme. This resulted in accumulation of intracellular cGMP and activation of cGMP-dependent protein kinase (PKG) at concentrations comparable to those that induced breast tumor cell apoptosis. Mirroring the effects on PKG, SSA also resulted in decreased Wnt/β-catenin signaling, including decreased expression of the Tcf/Lef controlled proteins, cyclin D1 and survivin. Furthermore, SSA caused a 50% reduction in the incidence and a 30% reduction in the multiplicity of mammary tumors using the MNU model of mammary carcinogenesis. Interestingly, mammary tumors induced by MNU displayed high levels of PDE5 expression. These findings further demonstrate the COX-independent nature of the anticancer activity of certain NSAIDs, provide additional evidence that PDE5 may serve as an effective target for breast cancer chemoprevention, and reveal the utility of chemically modifying the NSAIDs to remove COX inhibitory activity while enhancing anticancer activity in order to produce safer and more effective chemopreventive agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 570. doi:1538-7445.AM2012-570

Retinoic Acid Enhances Natriuretic Peptide Receptor‐A gene Transcription: Role of Additive Effect of Retinoic Acid Receptor‐α and Ets‐1

Activation of guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) by cardiac hormones atrial and brain natriuretic peptides produces the second messenger cGMP, which activates downstream signaling and biological effects of NPRA including vasorelaxation, anti‐mitogenic, and anti‐hypertrophic effects. The objective of the present study was to gain insight into the signaling mechanism of all‐trans retinoic acid (ATRA) in the regulation of Npr1 gene (coding for GC‐A/NPRA) transcription in cultured mouse mesangial cells. Cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and ITS (insulin,transferrin, and sodium selenite) and were transiently transfected using Lipofectamine. The results showed that ATRA significantly increased Npr1 mRNA, NPRA protein expression, and intracellular accumulation of cGMP in a dose‐dependent manner. ATRA caused a 7‐fold increase in luciferase activity from the region −365 to +55 base pairs of the Npr1 promoter. ATRA‐dependent Npr1 gene transcription was effectively inhibited by Ro 41‐5253, a specific antagonist for RARα. ATRA enhanced in vivo binding of Ets‐1 to Npr1 promoter as confirmed by ChIP assay. Moreover, sequential ChIP assay showed simultaneous presence of RARα and Ets‐1 in the Npr1 promoter containing Ets‐1 binding sites. Collectively, our results show that retinoic acid induces Npr1 gene transcription and expression via RARα and Ets‐1 transcription factors. The findings of the present studies will have important implications in prevention of high blood pressure and cardiac remodeling.