Inhibition of endogenous ouabain by atrial natriuretic peptide is a guanylyl cyclase independent effect.

BACKGROUND: Endogenous ouabain (EO) and atrial natriuretic peptide (ANP) are important in regulation of sodium and fluid balance. There is indirect evidence that ANP may be involved in the regulation of endogenous cardenolides. METHODS: H295R are human adrenocortical cells known to release EO. Cells were treated with ANP at physiologic concentrations or vehicle (0.1% DMSO), with or without guanylyl cyclase inhibitor 1,2,4 oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Cyclic guanosine monophosphate (cGMP), the intracellular second messenger of ANP, was measured by a chemiluminescent immunoassay and EO was measured by radioimmunoassay of C18 extracted samples. RESULTS: EO secretion is inhibited by ANP treatment, with the most prolonged inhibition (90 min vs ≤ 60 min) occurring at physiologic ANP concentrations (50 pg/mL). Inhibition of guanylyl cyclase with ODQ, also reduces EO secretion. The inhibitory effects on EO release in response to cotreatment with ANP and ODQ appeared to be additive. CONCLUSIONS: ANP inhibits basal EO secretion, and it is unlikely that this is mediated through ANP-A or ANP-B receptors (the most common natriuretic peptide receptors) or their cGMP second messenger; the underlying mechanisms involved are not revealed in the current studies. The role of ANP in the control of EO synthesis and secretion in vivo requires further investigation.

The cyclin dependent kinase inhibitor Roscovitine prevents diet-induced metabolic disruption in obese mice.

Most strategies to treat obesity-related disorders have involved prevention of diet-induced weight gain in lean mice. Treatment of obese individuals will require therapies that reverse the detrimental effects of excess body weight. Cyclin-dependent kinases have been shown to contribute to obesity and its adverse complications. Here, we show that roscovitine; a an orally available cyclin-dependent kinase inhibitor; given to male mice during the last six weeks of a 19-week high fat diet, reduced weight gain and prevented accompanying insulin resistance, hepatic steatosis, visceral adipose tissue (eWAT) inflammation/fibrosis as well as restored insulin secretion and enhanced whole body energy expenditure. Proteomics and phosphoproteomics analysis of eWAT demonstrated that roscovitine suppressed expression of peptides and phosphopeptides linked to inflammation and extracellular matrix proteins. It also identified 17 putative protein kinases perturbed by roscovitine, including CMGC kinases, AGC kinases and CAMK kinases. Pathway enrichment analysis showed that lipid metabolism, TCA cycle, fatty acid beta oxidation and creatine biosynthesis are enriched following roscovitine treatment. For brown adipose tissue (BAT), analysis of upstream kinases controlling the phosphoproteome revealed two major kinase groups, AGC and CMGC kinases. Among the top enriched pathways were insulin signaling, regulation of lipolysis in adipocytes, thyroid hormone signaling, thermogenesis and cAMP-PKG signaling. We conclude that roscovitine is effective at preventing prolonged diet-induced metabolic disruption and restoring mitochondrial activity in BAT and eWAT.

Exposure to dexamethasone modifies transcriptomic responses of free-living stages of Strongyloides stercoralis.

Hyperinfection and disseminated infection by the parasitic nematode Strongyloides stercoralis can be induced by iatrogenic administration of steroids and immunosuppression and lead to an elevated risk of mortality. Responses of free-living stages of S. stercoralis to the therapeutic corticosteroid dexamethasone (DXM) were investigated using RNA-seq transcriptomes of DXM-treated female and male worms. A total of 17,950 genes representing the transcriptome of these free-living adult stages were obtained, among which 199 and 263 were differentially expressed between DXM-treated females and DXM-treated males, respectively, compared with controls. According to Gene Ontology analysis, differentially expressed genes from DXM-treated females participate in developmental process, multicellular organismal process, cell differentiation, carbohydrate metabolic process and embryonic morphogenesis. Others are involved in signaling and signal transduction, including cAMP, cGMP-dependent protein kinase pathway, endocrine system, and thyroid hormone pathway, as based on Kyoto Encyclopedia of Genes and Genomes analysis. The novel findings warrant deeper investigation of the influence of DXM on growth and other pathways in this neglected tropical disease pathogen, particularly in a setting of autoimmune and/or allergic disease, which may require the clinical use of steroid-like hormones during latent or covert strongyloidiasis.

Prostaglandin E2 stimulates cAMP signaling and resensitizes human leukemia cells to glucocorticoid-induced cell death.

Glucocorticoid (GC) resistance remains a clinical challenge in pediatric acute lymphoblastic leukemia where response to GC is a reliable prognostic indicator. To identify GC resistance pathways, we conducted a genome-wide, survival-based, short hairpin RNA screen in murine T-cell acute lymphoblastic leukemia (T-ALL) cells. Genes identified in the screen interfere with cyclic adenosine monophosphate (cAMP) signaling and are underexpressed in GC-resistant or relapsed ALL patients. Silencing of the cAMP-activating Gnas gene interfered with GC-induced gene expression, resulting in dexamethasone resistance in vitro and in vivo. We demonstrate that cAMP signaling synergizes with dexamethasone to enhance cell death in GC-resistant human T-ALL cells. We find the E prostanoid receptor 4 expressed in T-ALL samples and demonstrate that prostaglandin E2 (PGE2) increases intracellular cAMP, potentiates GC-induced gene expression, and sensitizes human T-ALL samples to dexamethasone in vitro and in vivo. These findings identify PGE2 as a target for GC resensitization in relapsed pediatric T-ALL.

Dopamine suppresses osteoclast differentiation via cAMP/PKA/CREB pathway.

How the nervous system regulates bone remodeling is an exciting area of emerging research in bone biology. Accumulating evidence suggest that neurotransmitter-mediated inputs from neurons may act directly on osteoclasts. Dopamine is a neurotransmitter that can be released by hypothalamic neurons to regulate bone metabolism through the hypothalamic-pituitary-gonadal axis. Dopamine is also present in sympathetic nerves that penetrate skeletal structures throughout the body. It has been shown that dopamine suppresses osteoclast differentiation via a D2-like receptors (D2R)-dependent manner, but the intracellular secondary signaling pathway has not been elucidated. In this study, we found that cAMP-response element binding protein (CREB) activity responds to dopamine treatment during osteoclastogenesis. Considering the critical role of CREB in osteoclastogenesis, we hypothesize that CREB may be a critical target in dopamine's regulation of osteoclast differentiation. We confirmed that D2R is also present in RAW cells and activated by dopamine. Binding of dopamine to D2R inhibits the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling pathway which ultimately decreases CREB phosphorylation during osteoclastogenesis. This was also associated with diminished expression of osteoclast markers that are downstream of CREB. Pharmacological activation of adenylate cyclase (to increase cAMP production) and PKA reverses the effect of dopamine on CREB activity and osteoclastogenesis. Therefore, we have identified D2R/cAMP/PKA/CREB as a candidate pathway that mediates dopamine's inhibition of osteoclast differentiation. These findings will contribute to our understanding of how the nervous and skeletal systems interact to regulate bone remodeling. This will enable future work toward elucidating the role of the nervous system in bone development, repair, aging, and degenerative disease.

A 5-mer peptide derived from hinge region of hFSHR can function as positive allosteric modulator in vivo.

Interaction of follicle stimulating hormone (FSH) with its cognate receptor (FSHR) is critical for maintaining reproductive health. FSHR has a large extracellular domain (ECD), composed of leucine rich repeats (LRRs) and hinge region, a transmembrane domain (TMD) and a short C-terminal domain (CTD). In this study, we have identified a short peptidic stretch in the hinge region (hFSHR(271-275)), through extensive computational modeling, docking and MD simulations, that is capable of independently interacting with the extracellular loops of FSHR(TMD). In vitro studies revealed that FSHR(271-275) peptide increased binding of [125I]-FSH to rat Fshr as well as FSH-induced cAMP production. Administration of FSHR(271-275) peptide in immature female rats significantly increased FSH-mediated ovarian weight gain and promoted granulosa cell proliferation. In summary, the results demonstrate that the synthetic peptide corresponding to amino acids 271-275 of hFSHR-hinge region stimulates FSH-FSHR interaction and behaves as positive allosteric modulator of FSHR. The study also lends evidence to the existing proposition that hinge region maintains the receptor in an inactive conformation in the absence of its ligand by engaging in intramolecular interactions with extracellular loops of TMD.

Influence of Phosphodiesterase Inhibition on CRE- and EGR1-Dependent Transcription in a Mouse Hippocampal Cell Line.

Signaling pathways, depending on the second messenger molecule cAMP, modulate hippocampal cell signaling via influencing transcription factors like cAMP-regulated element-binding protein (CREB) or early growth response 1 EGR1/Krox24/zif268/ZENK (EGR1). Here, we investigated two reporter cell lines derived from an immortalized hippocampal neuronal cell line stably expressing a CRE- or EGR1-luciferase reporter gene (HT22CREluc and HT22EGR1luc, respectively). The cells were subjected to phosphodiesterase inhibitors and other cAMP-modulating agents to investigate dose- and time-dependent phosphodiesterase (PDE)-mediated fine-tuning of cAMP-dependent transcriptional signaling. The non-isoform-specific cyclic nucleotide phosphodiesterase (PDE) inhibitor isobutyl-methyl-xanthine (IBMX), as well as selective inhibitors of PDE3 (milrinone) and PDE4 (rolipram), were tested for their ability to elevate CRE- and EGR1-luciferase activity. Pharmacological parameters like onset of activity, maximum activity, and offset of activity were determined. In summary, phosphodiesterase inhibition appeared similarly potent in comparison to adenylate cyclase stimulation or direct activation of protein kinase A (PKA) via specific cAMP agonists and was at least partly mediated by PKA as shown by the selective PKA inhibitor Rp-8-Br-cAMPS. Moreover, transcriptional activation by PDE inhibition was also influenced by organic anion-exchanger action and interacted with fibroblast growth factor (FGF) receptor-mediated pathways.

Adenylate Cyclase Toxin Tinkering With Monocyte-Macrophage Differentiation.

Circulating inflammatory monocytes are attracted to infected mucosa and differentiate into macrophage or dendritic cells endowed with enhanced bactericidal and antigen presenting capacities. In this brief Perspective we discuss the newly emerging insight into how the cAMP signaling capacity of Bordetella pertussis adenylate cyclase toxin manipulates the differentiation of monocytes and trigger dedifferentiation of the alveolar macrophages to facilitate bacterial colonization of human airways.

Extracellular ATP modulates podocyte function through P2Y purinergic receptors and pleiotropic effects on AMPK and cAMP/PKA signaling pathways.

Podocytes and their foot processes interlinked by slit diaphragms, constitute a continuous outermost layer of the glomerular capillary and seem to be crucial for maintaining the integrity of the glomerular filtration barrier. Purinergic signaling is involved in a wide range of physiological processes in the renal system, including regulating glomerular filtration. We evaluated the role of nucleotide receptors in cultured rat podocytes using non-selective P2 receptor agonists and agonists specific for the P2Y1, P2Y2, and P2Y4 receptors. The results showed that extracellular ATP evokes cAMP-dependent pathways through P2 receptors and influences remodeling of the podocyte cytoskeleton and podocyte permeability to albumin via coupling with RhoA signaling. Our findings highlight the relevance of the P2Y4 receptor in protein kinase A-mediated signal transduction to the actin cytoskeleton. We observed increased cAMP concentration and decreased RhoA activity after treatment with a P2Y4 agonist. Moreover, protein kinase A inhibitors reversed P2Y4-induced changes in RhoA activity and intracellular F-actin staining. P2Y4 stimulation resulted in enhanced AMPK phosphorylation and reduced reactive oxygen species generation. Our findings identify P2Y-PKA-RhoA signaling as the regulatory mechanism of the podocyte contractile apparatus and glomerular filtration. We describe a protection mechanism for the glomerular barrier linked to reduced oxidative stress and reestablished energy balance.

A Tissue-Engineered Human Psoriatic Skin Model to Investigate the Implication of cAMP in Psoriasis: Differential Impacts of Cholera Toxin and Isoproterenol on cAMP Levels of the Epidermis.

Pathological and healthy skin models were reconstructed using similar culture conditions according to well-known tissue engineering protocols. For both models, cyclic nucleotide enhancers were used as additives to promote keratinocytes' proliferation. Cholera toxin (CT) and isoproterenol (ISO), a beta-adrenergic agonist, are the most common cAMP stimulators recommended for cell culture. The aim of this study was to evaluate the impact of either CT or ISO on the pathological characteristics of the dermatosis while producing a psoriatic skin model. Healthy and psoriatic skin substitutes were produced according to the self-assembly method of tissue engineering, using culture media supplemented with either CT (10-10 M) or ISO (10-6 M). Psoriatic substitutes produced with CT exhibited a more pronounced psoriatic phenotype than those produced with ISO. Indeed, the psoriatic substitutes produced with CT had the thickest epidermis, as well as contained the most proliferating cells and the most altered expression of involucrin, filaggrin, and keratin 10. Of the four conditions under study, psoriatic substitutes produced with CT had the highest levels of cAMP and enhanced expression of adenylate cyclase 9. Taken together, these results suggest that high levels of cAMP are linked to a stronger psoriatic phenotype.

Phospholipid Signaling Is a Component of the Salicylic Acid Response in Plant Cell Suspension Cultures.

Salicylic acid (SA) is an important signaling molecule involved in plant defense. While many proteins play essential roles in SA signaling, increasing evidence shows that responses to SA appear to involve and require lipid signals. The phospholipid-generated signal transduction involves a family of enzymes that catalyze the hydrolysis or phosphorylation of phospholipids in membranes to generate signaling molecules, which are important in the plant cellular response. In this review, we focus first, the role of SA as a mitigator in biotic/abiotic stress. Later, we describe the experimental evidence supporting the phospholipid-SA connection in plant cells, emphasizing the roles of the secondary lipid messengers (phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA)) and related enzymes (phospholipase D (PLD) and phospholipase C (PLC)). By placing these recent finding in context of phospholipids and SA in plant cells, we highlight the role of phospholipids as modulators in the early steps of SA triggered transduction in plant cells.

cGMP Signaling in Cardiovascular Diseases: Linking Genotype and Phenotype.

Cyclic guanosine 3',5'-monophosphate (cGMP) is the key second messenger molecule in nitric oxide signaling. Its rapid generation and fate, but also its role in mediating acute cellular functions has been extensively studied. In the past years, genetic studies suggested an important role for cGMP in affecting the risk of chronic cardiovascular diseases, for example, coronary artery disease and myocardial infarction. Here, we review the role of cGMP in atherosclerosis and other cardiovascular diseases and discuss recent genetic findings and identified mechanisms. Finally, we highlight open questions and promising research topics.

Glucagon-like peptide 1 reverses myocardial hypertrophy through cAMP/PKA/RhoA/ROCK2 signaling.

Myocardial hypertrophy is a major pathological and physiological process during heart failure. Glucagon-like peptide 1 (GLP-1) is a glucagon incretin hormone released from the gut endocrine L-cells that has protective effects on various cardiovascular diseases, including hypertension, atherosclerosis, and myocardial hypertrophy. However, the protective mechanisms of GLP-1 in myocardial hypertrophy remain unclear. Here, we showed that the GLP-1 agonist liraglutide and dipeptidyl peptidase 4 inhibitor alogliptin decreased heart weight and cardiac muscle cell volume in spontaneously hypertensive rats (SHR). In H9C2 cell hypertensive models induced by angiotensin II, GLP-1 treatment reduced myocardial cell volume, inhibited the expressions of atrial natriuretic peptide, brain/B-type natriuretic peptide, ß-myosin heavy chain, RhoA, and ROCK2, and decreased MLC and MYPT1 phosphorylation. When H9C2 cells were treated with H89, a PKA inhibitor, the inhibitory effect of GLP-1 disappeared, while the inhibitory role was enhanced under the treatment of Y-27632, a ROCK2 inhibitor. These results suggested that GLP-1 might reverse myocardial hypertrophy through the PKA/RhoA/ROCK2 signaling pathway.

Argania Spinosa Fruit Shell Extract-Induced Melanogenesis via cAMP Signaling Pathway Activation.

We have previously reported that argan oil and argan press-cake from the kernels of Argania spinosa have an anti-melanogenesis effect. Here, the effect of argan fruit shell ethanol extract (AFSEE) on melanogenesis in B16F10 cells was determined, and the mechanism underlying its effect was elucidated. The proliferation of AFSEE-treated B16F10 cells was evaluated using the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay, while the melanin content was quantified using a spectrophotometric method. The expression of melanogenesis-related proteins was determined by Western blot and real-time PCR, while global gene expression was determined using a DNA microarray. In vitro analysis results showed that the melanin content of B16F10 cells was significantly increased by AFSEE, without cytotoxicity, by increasing the melanogenic enzyme tyrosinase (TRY), tyrosinase related-protein 1 (TRP1), and dopachrome tautomerase (DCT) protein and mRNA expression, as well as upregulating microphthalmia-associated transcription factor (MITF) expression through mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK) and p38, and the cyclic adenosine monophosphate (cAMP) signaling pathway, as indicated by the microarray analysis results. AFSEE's melanogenesis promotion effect is primarily attributed to its polyphenolic components. In conclusion, AFSEE promotes melanogenesis in B16F10 cells by upregulating the expression of the melanogenic enzymes through the cAMP-MITF signaling pathway.AFSEE may be used as a cosmetics product component to promote melanogenesis, or as a therapeutic against hypopigmentation disorders.

Targets of cGMP/cGKI in Cardiac Myocytes.

The 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase type I (cGKI aka PKGI) is a major cardiac effector acting downstream of nitric oxide (NO)-sensitive soluble guanylyl cyclase and natriuretic peptides (NPs), which signal through transmembrane guanylyl cyclases. Consistent with the wide distribution of the cGMP-generating guanylyl cyclases, cGKI, which usually elicits its cellular effects by direct phosphorylation of its targets, is present in multiple cardiac cell types including cardiomyocytes (CMs). Although numerous targets of cGMP/cGKI in heart were identified in the past, neither their exact patho-/physiological functions nor cell-type specific roles are clear. Herein, we inform about the current knowledge on the signal transduction downstream of CM cGKI. We believe that better insights into the specific actions of cGMP and cGKI in these cells will help to guide future studies in the search for predictive biomarkers for the response to pharmacological cGMP pathway modulation. In addition, targets downstream of cGMP/cGKI may be exploited for refined and optimized diagnostic and therapeutic strategies in different types of heart disease and their causes. Importantly, key functions of these proteins and particularly sites of regulatory phosphorylation by cGKI should, at least in principle, remain intact, although upstream signaling through the second messenger cGMP is impaired or dysregulated in a stressed or diseased heart state.

Magnesium Acts as a Second Messenger in the Regulation of NMDA Receptor-Mediated CREB Signaling in Neurons.

Extracellular magnesium ion ([Mg2+]) is a well-known voltage-dependent blocker of NMDA receptors, which plays a critical role in the regulation of neuronal plasticity, learning, and memory. It is generally believed that NMDA receptor activation involves in Mg2+ being removed into extracellular compartment from the channel pore. On the other hand, Mg2+ is one of the most abundant intracellular cations, and involved in numerous cellular functions. However, we do not know if extracellular magnesium ions can influx into neurons to affect intracellular signaling pathways. In our current study, we found that extracellular [Mg2+] elevation enhanced CREB activation by NMDA receptor signaling in both mixed sex rat cultured neurons and brain slices. Moreover, we found that extracellular [Mg2+] led to CREB activation by NMDA application, albeit in a delayed manner, even in the absence of extracellular calcium, suggesting a potential independent role of magnesium in CREB activation. Consistent with this, we found that NMDA application leads to an NMDAR-dependent increase in intracellular-free [Mg2+] in cultured neurons in the absence of extracellular calcium. Chelating this magnesium influx or inhibiting P38 mitogen-activated protein kinase (p38 MAPK) blocked the delayed pCREB by NMDA. Finally, we found that NMDAR signaling in the absence of extracellular calcium activates p38 MAPK. Our studies thus indicate that magnesium influx, dependent on NMDA receptor opening, can transduce a signaling pathway to activate CREB in neurons.

Glucocorticoids rapidly activate cAMP production via Gαs to initiate non-genomic signaling that contributes to one-third of their canonical genomic effects.

Glucocorticoids are widely used for the suppression of inflammation, but evidence is growing that they can have rapid, non-genomic actions that have been unappreciated. Diverse cell signaling effects have been reported for glucocorticoids, leading us to hypothesize that glucocorticoids alone can swiftly increase the 3',5'-cyclic adenosine monophosphate (cAMP) production. We found that prednisone, fluticasone, budesonide, and progesterone each increased cAMP levels within 3 minutes without phosphodiesterase inhibitors by measuring real-time cAMP dynamics using the cAMP difference detector in situ assay in a variety of immortalized cell lines and primary human airway smooth muscle (HASM) cells. A membrane- impermeable glucocorticoid showed similarly rapid stimulation of cAMP, implying that responses are initiated at the cell surface. siRNA knockdown of Gαs virtually eliminated glucocorticoid-stimulated cAMP responses, suggesting that these drugs activate the cAMP production via a G protein-coupled receptor. Estradiol had small effects on cAMP levels but G protein estrogen receptor antagonists had little effect on responses to any of the glucocorticoids tested. The genomic and non-genomic actions of budesonide were analyzed by RNA-Seq analysis of 24 hours treated HASM, with and without knockdown of Gαs . A 140-gene budesonide signature was identified, of which 48 genes represent a non-genomic signature that requires Gαs signaling. Collectively, this non-genomic cAMP signaling modality contributes to one-third of the gene expression changes induced by glucocorticoid treatment and shifts the view of how this important class of drugs exerts its effects.

cGMP Signaling and Modulation in Heart Failure.

Cyclic GMP (cGMP) represents a classic intracellular second messenger molecule. Over the past 2 decades, important discoveries have identified that cGMP signaling becomes deranged in heart failure (HF) and that cGMP and its main kinase effector, protein kinase G, generally oppose the biological abnormalities contributing to HF, in experimental studies. These findings have influenced the design of clinical trials of cGMP-augmenting drugs in HF patients. At present, the trial results of cGMP-augmenting therapies in HF remain mixed. As detailed in this review, strong evidence now exists that protein kinase G opposes pathologic cardiac remodeling through regulation of diverse biological processes and myocardial substrates. Potential reasons for the failures of cGMP-augmenting drugs in HF may be related to biological mechanisms opposing cGMP or because of certain features of clinical trials, all of which are discussed.

Multiplicity of Nitric Oxide and Natriuretic Peptide Signaling in Heart Failure.

Heart failure (HF) is a common consequence of several cardiovascular diseases and is understood as a vicious cycle of cardiac and hemodynamic decline. The current inventory of treatments either alleviates the pathophysiological features (eg, cardiac dysfunction, neurohumoral activation, and ventricular remodeling) and/or targets any underlying pathologies (eg, hypertension and myocardial infarction). Yet, since these do not provide a cure, the morbidity and mortality associated with HF remains high. Therefore, the disease constitutes an unmet medical need, and novel therapies are desperately needed. Cyclic guanosine-3',5'-monophosphate (cGMP), synthesized by nitric oxide (NO)- and natriuretic peptide (NP)-responsive guanylyl cyclase (GC) enzymes, exerts numerous protective effects on cardiac contractility, hypertrophy, fibrosis, and apoptosis. Impaired cGMP signaling, which can occur after GC deactivation and the upregulation of cyclic nucleotide-hydrolyzing phosphodiesterases (PDEs), promotes cardiac dysfunction. In this study, we review the role that NO/cGMP and NP/cGMP signaling plays in HF. After considering disease etiology, the physiological effects of cGMP in the heart are discussed. We then assess the evidence from preclinical models and patients that compromised cGMP signaling contributes to the HF phenotype. Finally, the potential of pharmacologically harnessing cardioprotective cGMP to rectify the present paucity of effective HF treatments is examined.