Increase In cAMP Concentration Research Articles

Linoleic acid enhance the production of moncolin K and red pigments in Monascus ruber by activating mokH and mokA, and by accelerating cAMP-PkA pathway

Monacolin K, an inhibitor of HMG-CoA reductase, is a secondary metabolite synthesized by polyketide synthases (PKS) from Monascus ruber. The mokH gene encoding Zn(II)2Cys6 binding protein and mokA gene encoding polyketide synthase are presumed to activate monacolin K production. In this study, linoleic acid could be a quorum sensing signaling molecule to increase monacolin K production in the cyclic AMP(cAMP)—protein kinase A(PKA) signaling pathway. Analysis of the PKA activity and the cAMP concentration shows that linoleic acid could increase cAMP concentration and activate PKA. Analysis of the RT-qPCR products demonstrates that 256μM and 512μM linoleic acid can up-regulate mokH and mokA gene transcript levels. Especially with 512μM linoleic acid addition, linoleic acid increase 1.35 folds of monacolin K production, but 64μM linoleic acid increase 1.94 folds of red pigment production in Monascus ruber. These results show the cAMP-PkA pathway activity can up-regulate mokA and mokH gene, which enhance the yield of Monacolin K.

Evaluation of the insulinotropic and glucose-lowering actions of zebrafish GIP in mammalian systems: Evidence for involvement of the GLP-1 receptor

The insulinotropic properties of zebrafish GIP (zfGIP) were assessed in vitro using clonal pancreatic β-cell lines and isolated mouse islets and acute effects on glucose tolerance and insulin release in vivo were evaluated in mice. The peptide produced a dose-dependent increase in the rate of insulin release from BRIN-BD11 rat clonal β-cells at concentrations ≥30nM. Insulin release from 1.1 B4 human clonal β-cells and mouse islets was significantly increased by zfGIP (10nM and 1μM). The in vitro insulinotropic activity of zfGIP was decreased after incubating BRIN-BD11 cells with the GLP-1 receptor antagonist, exendin-4(9-39) (p<0.001) and the GIP receptor antagonist, GIP (6-30) Cex-K40[Pal] (p<0.05) but the glucagon receptor antagonist [des-His1,Pro4,Glu9]glucagon amide was without effect. zfGIP (10nM and 1μM) produced significant increases in cAMP concentration in CHL cells transfected with the human GLP-1 receptor but was without effect on HEK293 cells transfected with the human glucagon receptor. Conversely, zfGIP, but not human GIP, significantly stimulated insulin release from CRISPR/Cas9-engineered INS-1 clonal β-cells from which the GIP receptor had been deleted. Intraperitoneal administration of zfGIP (25 and 75nmol/kg body weight) to mice together with an intraperitoneal glucose load (18mmol/kg body weight) produced a significant decrease in plasma glucose concentrations concomitant with an increase in insulin concentrations. The study provides evidence that the insulinotropic action of zfGIP in mammalian systems involves activation of both the GLP-1 and the GIP receptors but not the glucagon receptor.

Heptamethoxyflavone Reduces Phosphodiesterase Activity and T-Cell Growth in vitro

In a previous study, we reported that interleukin-4 production was reduced in spleen cells of mice administered 3,5,6,7,8,3′,4′-heptamethoxyflavone (HMF), which is a polymethoxyflavone found at high concentrations in the peel of various citrus fruits. In this study, we investigated the function of HMF on the growth of T cells cultured from the spleens of mice. HMF decreased the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by anti-CD3/CD28 antibody-stimulated mouse spleen cells. HMF inhibited the activities of phosphodiesterase (PDE) enzymes prepared from bovine brain and human PDE_4B and PDE_3B enzymes. The cyclic AMP (cAMP) content in anti-CD3/CD28 antibody-stimulated spleen cells increased after HMF treatment in vitro. These results suggest that HMF inhibits T-cell growth and affects immune function via reduced PDE activity and increased cAMP content.

Identification of adenylyl cyclase isoforms mediating parathyroid hormone- and calcitonin-stimulated cyclic AMP accumulation in distal tubule cells

BackgroundThe distal convoluted tubule (DCT) is an important nephron site for parathyroid hormone (PTH) and calcitonin regulation of urinary divalent cation excretion. These hormones exert their effects on the DCT in substantial part through activation of adenylyl cyclase (AC); however, it is unknown which AC isoforms are involved.MethodsTo examine this, two mouse DCT cell lines were studied: 209 and D1 cells. AC isoform mRNA expression was analyzed by real-time PCR. Cyclic AMP was measured using enzyme immunoassay.ResultsCalcitonin, but not PTH, stimulated cAMP accumulation in 209 cells, while PTH, but not calcitonin, increased cAMP content in D1 cells. Both cell types expressed AC3, AC4, AC6, AC7, and AC9 mRNA; in both cell types, AC6 mRNA was most abundant, followed by AC9, then AC3 and AC7, with relatively very small amounts of AC4 mRNA. Microdissected mouse DCT had a similar pattern of AC isoform mRNA expression although AC5 mRNA was detected. Individual siRNA knockdown of AC6 and AC9 reduced calcitonin-stimulated cAMP accumulation in 209 cells and PTH-induced cAMP levels in D1 cells. Knockdown of AC3 had no effect on hormonal augmentation of cAMP in either cell line. Surprisingly, knockdown of AC7 increased calcitonin-induced cAMP accumulation in 209 cells as well as PTH-stimulated cAMP content in D1 cells.ConclusionsTaken together, these findings indicate that AC6 and AC9 mediate calcitonin- and PTH-stimulated cAMP accumulation in DCT cells, while activation of AC7 may paradoxically reduce the stimulatory effects of PTH and calcitonin on cultured DCT cAMP levels.

Modulation of epithelial sodium channel in human alveolar epithelial cells by lipoxin A4 through AhR-cAMP-dependent pathway

Purpose: To investigate the effect of lipoxin A4 (LXA4) on the expressions of protein and mRNA of alveolar epithelial sodium channel (ENaC) in normal and lipopolysaccharide (LPS)-stimulated A549 cells. Methods: A549 cell-lines were randomized into 11 groups ( N = 8) and treated. EnaC level was evaluated by Western blot. Total RNA was extracted and reverse-transcribed and then levels of ENaC mRNA, cGMP and cAMP in the cells were determined. Results: LXA4 (10 -7 mol/L) increased the expressions of α-subunit of ENaC relative to LPS group. In addition, LXA4 significantly up-regulated the expression of mRNAs of α, β and γ subunits of ENaC ( p < 0.01). The level of cAMP was increased in LXA4 group, but significantly reduced in LPS group relative to control group ( p < 0.05). However, treatment with LXA4 annulled the increased cAMP concentration, compared with LPS group ( p < 0.05) Conclusion: These results show that LXA4 influences ENaC up-regulation in normal and LPS stimulated A549 alveolar epithelial cells. Keywords: Acute lung injury, Alveolar epithelial sodium channel, Lipoxin A4, AhR, cAMP, cGMP

Abstract P240: Decarboxylation of Ang-(1-7) to Ala 1 -ang-(1-7) Leads to Major Changes in Pharmacodynamics

Background: Within the renin-angiotensin system, angiotensin (Ang)-(1-7) is cardiovascular protective, stimulates regeneration, and opposes the often detrimental effects of Ang II. We identified two receptors for the heptapeptide; the G protein-coupled receptors Mas and MrgD. Recently, a decarboxylated form of Ang-(1-7), Ala 1 -Ang-(1-7) (Alamandine), has been described as having similar vasorelaxing effects as Ang-(1-7) but distinctively stimulating the MrgD receptor. Methods and Results: The aim of this study was to elucidate the consequences of the lack in the carboxyl group in amino acid one on intracellular signalling, to discover the receptor fingerprint for Ala 1 -Ang-(1-7), and to characterize the consequences for pharmacodynamics. Therfore cAMP was used as the main readout in receptor-transfected HEK293 cells as well as primary cells (mesangial cells and HUVEC). Ala 1 -Ang-(1-7) elevated cAMP concentrations in primary endothelial and mesangial cells. However, the dose-response curves clearly discriminated from the curves generated with Ang-(1-7) (EC 50 = 1.1 x 10 -8 M), with much lower EC 50 (EC 50 = 3.6 x 10 -11 M) and bell-shape for Ala 1 -Ang-(1-7). We provided pharmacological proof that both, Mas (EC 50 = 6.31 x 10 -12 M) and MrgD (EC 50 = 3.98 x 10 -13 M), are functional receptors for Ala 1 -Ang-(1-7). Consequently, the heptapeptide failed to increase cAMP concentration in primary mesangial cells with genetic deficiency in both receptors. As for Ang-(1-7), the AT2 blocker PD123319 also blocked the Ala 1 -Ang-(1-7) effects on Mas and MrgD receptors and in primary cells. The very distinct dose-response curves for both heptapeptides could be explained by in silico modelling, energy calculations, and an involvement of G alpha i for higher concentrations of Ala 1 -Ang-(1-7). Conclusions: Our results identify Ala 1 -Ang-(1-7) as a peptide with specific pharmacodynamic properties and build the basis for the design of more potent and efficient Ang-(1-7) analogues for therapeutic interventions in a rapidly growing number of diseases.

Cannabinoid CB1 receptor inverse agonist MJ08 stimulates glucose production via hepatic sympathetic innervation in rats

As a key insulin target tissue for maintaining systemic glucose homeostasis, the liver plays important roles in improving obesity-associated insulin intolerance via selective cannabinoid CB1 receptor antagonism/inverse agonism. However, it is unclear whether this receptor inverse agonism affects hepatic glucose metabolism. MJ08 is a novel cannabinoid CB1 receptor antagonist/inverse agonist that has superior inverse agonism over the well-known antagonist/inverse agonist, SR141716 (rimonabant). MJ08 remarkably elevates fasting blood glucose independent of inhibition of insulin release in mice. In the current study, MJ08 was used to investigate the mechanism by which liver cannabinoid CB1 receptor inverse activation regulates hepatic glucose metabolism. MJ08 stimulated hepatic glucose production (HGP) in a dose-dependent manner and promoted gluconeogenic gene expression in perfused rat liver. SR141716 exhibited similar but weaker effects. The cannabinoid CB1 receptor agonist (WIN 55,212-2), Gs protein-cyclic AMP (cAMP)-dependent pathway inhibitors (NF449 and H89), β-adrenoceptor antagonist (propranolol), and peripheral sympathetic inhibitor (reserpine) could antagonize MJ08-induced HGP. Furthermore, MJ08 and SR141716 induced monoamine neurotransmitter (noradrenaline) release and increased cAMP content significantly in perfused liver, although only a slight increase was observed in primary cultured hepatocytes. These results indicate that local liver cannabinoid CB1 receptor inverse agonism via hepatic sympathetic innervation is responsible for the HGP induced by MJ08. Thus, high inverse agonistic activity could increase fasting blood glucose levels and should be avoided in the development of peripheral cannabinoid CB1 receptor-targeted weight-loss drugs.

A low [Ca2+]i-induced enhancement of cAMP-activated ciliary beating by PDE1A inhibition in mouse airway cilia.

This study demonstrated that PDE1 (phosphodiesterase 1) existing in the ciliary beat frequency (CBF)-regulating metabolon regulates CBF in procaterol-stimulated lung airway ciliary cells of mouse. Procaterol (an β2-agonist) increased the ciliary bend angle (CBA) and CBF via cAMP accumulation in the ciliary cells of mice: interestingly, the time course of CBF increase was slower than that of CBA increase. However, IBMX (3-isobutyl-1-methylxanthine, an inhibitor of PDE) increased CBA and CBF in an identical time course. Lowering an intracellular Ca2+ concentration ([Ca2+]i) caused by switching to an EGTA-containing Ca2+-free solution from normal one elevated the procaterol-induced increasing rate of CBF. These observations suggest that Ca2+-dependent PDE1 controls cAMP-stimulated CBF increase. Either application of 8MmIBMX (8-methoxymethyl-IBMX, a selective PDE1 inhibitor), BAPTA-AM (an intracellular Ca2+ chelator), or calmidazolium (an inhibitior of calmodulin) alone increased CBA and CBF in the lung airway ciliary cells and increased cAMP contents in the isolated lung cells, and like IBMX, each application of the compound made the time courses of CBA and CBF increase stimulated by procaterol identical. The immunoelectron microscopic examinations revealed that PDE1A exists in the space between the nine doublet tubules ring and plasma membrane in the lung airway cilium, where the outer dynein arm (a molecular motor regulating CBF) functions. In conclusion, PDE1A is a key factor slowing the time course of the procaterol-induced increase in CBF via degradation of cAMP in the CBF-regulating metabolon of the mouse lung airway cilia.

Actions of PGLa-AM1 and its [A14K] and [A20K] analogues and their therapeutic potential as anti-diabetic agents

PGLa-AM1 (GMASKAGSVL10GKVAKVALKA20AL.NH2) was first identified in skin secretions of the frog Xenopus amieti (Pipidae) on the basis of its antimicrobial properties. PGLa-AM1 and its [A14K] and [A20K] analogues produced a concentration-dependent stimulation of insulin release from BRIN-BD11 rat clonal β-cells without cytotoxicity at concentrations up to 3 μM. In contrast, the [A3K] analogue was cytotoxic at concentrations ≥ 30 nM. The potency and maximum rate of insulin release produced by the [A14K] and [A20K] peptides were significantly greater than produced by PGLa-AM1. [A14K]PGLa-AM1 also stimulated insulin release from mouse islets at concentrations ≥ 1 nM and from the 1.1B4 human-derived pancreatic β-cell line at concentrations > 30 pM. PGLa-AM1 (1 μM) produced membrane depolarization in BRIN-BD11 cells with a small, but significant (P < 0.05), increase in intracellular Ca2+ concentrations but the peptide had no direct effect on KATP channels. The [A14K] analogue (1 μM) produced a significant increase in cAMP concentration in BRIN-BD11 cells and down-regulation of the protein kinase A pathway by overnight incubation with forskolin completely abolished the insulin-releasing effects of the peptide. [A14K]PGLa-AM1 (1 μM) protected against cytokine-induced apoptosis (p < 0.001) in BRIN-BD11 cells and augmented (p < 0.001) proliferation of the cells to a similar extent as GLP-1. Intraperitoneal administration of the [A14K] and [A20K] analogues (75 nmol/kg body weight) to both lean mice and high fat-fed mice with insulin resistance improved glucose tolerance with a concomitant increase in insulin secretion. The data provide further support for the assertion that host defense peptides from frogs belonging to the Pipidae family show potential for development into agents for the treatment of patients with Type 2 diabetes.

Screening with an NMNAT2-MSD platform identifies small molecules that modulate NMNAT2 levels in cortical neurons

Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) is a key neuronal maintenance factor and provides potent neuroprotection in numerous preclinical models of neurological disorders. NMNAT2 is significantly reduced in Alzheimer’s, Huntington’s, Parkinson’s diseases. Here we developed a Meso Scale Discovery (MSD)-based screening platform to quantify endogenous NMNAT2 in cortical neurons. The high sensitivity and large dynamic range of this NMNAT2-MSD platform allowed us to screen the Sigma LOPAC library consisting of 1280 compounds. This library had a 2.89% hit rate, with 24 NMNAT2 positive and 13 negative modulators identified. Western analysis was conducted to validate and determine the dose-dependency of identified modulators. Caffeine, one identified NMNAT2 positive-modulator, when systemically administered restored NMNAT2 expression in rTg4510 tauopathy mice to normal levels. We confirmed in a cell culture model that four selected positive-modulators exerted NMNAT2-specific neuroprotection against vincristine-induced cell death while four selected NMNAT2 negative modulators reduced neuronal viability in an NMNAT2-dependent manner. Many of the identified NMNAT2 positive modulators are predicted to increase cAMP concentration, suggesting that neuronal NMNAT2 levels are tightly regulated by cAMP signaling. Taken together, our findings indicate that the NMNAT2-MSD platform provides a sensitive phenotypic screen to detect NMNAT2 in neurons.

Effector Translocation: Cya Reporter Assay.

An accurate and complete roster of the Type III effector (T3E) proteins translocated by the P. syringae Type III secretion system (T3SS) into host cells is critical to understanding the pathogen's interactions with plants. The adenylate cyclase (Cya) reporter offers a highly sensitive and robust assay for monitoring the translocation of T3Es. T3Es are fused to the calmodulin-dependent adenylate-cyclase domain of CyaA. The T3E targets Cya for translocation through the T3SS into the host cell at which point it is activated by calmodulin and converts adenosine triphosphate into cyclic adenosine monophosphate (cAMP). The T3SS translocation-dependent increase in cAMP concentration in plant cells is then measured with an enzyme-linked immunosorbent assay kit. The Cya reporter can be used to determine whether a candidate protein is translocated by T3SS or to measure relative levels of T3SS translocation in a semiquantitative manner.

Naringenin Regulates CFTR Activation and Expression in Airway Epithelial Cells

Background/Aims: Sputum symptoms are commonly seen in the elderly. This study aimed to identify an efficacious expectorant treatment stratagem through evaluating the secretion-promoting activation and cystic fibrosis transmembrane conductance regulator (CFTR) expression of the bioactive herbal monomer naringenin. Methods: Vectorial Cl^- transport was determined by measuring short-circuit current (I_SC) in rat airway epithelium. cAMP content was measured by ELISA in primary cultured epithelial cells and Calu-3 cells. CFTR expression in Calu-3 cells was determined by qPCR. Results: Addition of naringenin to the basolateral side of the rat airway led to a concentration-dependent sustained increase in I_SC. The current was suppressed when exposed to Cl^–-free solution or by bumetanide, BaCl₂, and DPC but not by DIDS and IBMX. Forskolin-induced I_SC increase and CFTRinh-172/MDL-12330A-induced I_SC inhibition were not altered by naringenin. Intracellular cAMP content was significantly increased by naringenin. With lipopolysaccharide stimulation, CFTR expression was significantly reduced, and naringenin dose-dependently enhanced CFTR mRNA expression. Conclusion: These results demonstrate that naringenin has the ability to stimulate Cl^- secretion, which is mediated by CFTR through a signaling pathway by increasing cAMP content. Moreover, naringenin can increase CFTR expression when organism CFTR expression is seriously hampered. Our data suggest a potentially effective treatment strategy for sputum.

Dopamine directly increases mitochondrial mass and thermogenesis in brown adipocytes.

Brown adipose tissue (BAT) is key to energy homeostasis. By virtue of its thermogenic potential, it may dissipate excessive energy, regulate body weight and increase insulin sensitivity. Catecholamines are critically involved in the regulation of BAT thermogenesis, yet research has focussed on the effects of noradrenaline and adrenaline. Some evidence suggests a role of dopamine (DA) in BAT thermogenesis, but the cellular mechanisms involved have not been addressed. We employed our extensively characterised murine brown adipocyte cells. D1-like and D2-like receptors were detectable at the protein level. Stimulation with DA caused an increase in cAMP concentrations. Oxygen consumption rates (OCR), mitochondrial membrane potential (Δψm) and uncoupling protein 1 (UCP1) levels increased after 24 h of treatment with either DA or a D1-like specific receptor agonist. A D1-like receptor antagonist abolished the DA-mediated effect on OCR, Δψm and UCP1. DA induced the release of fatty acids, which did not additionally alter DA-mediated increases of OCR. Mitochondrial mass (as determined by (i) CCCP- and oligomycin-mediated effects on OCR and (ii) immunoblot analysis of mitochondrial proteins) also increased within 24 h. This was accompanied by an increase in peroxisome proliferator-activated receptor gamma co-activator 1 alpha protein levels. Also, DA caused an increase in p38 MAPK phosphorylation and pharmacological inhibition of p38 MAPK abolished the DA-mediated effect on Δψm In summary, our study is the first to reveal direct D1-like receptor and p38 MAPK-mediated increases of thermogenesis and mitochondrial mass in brown adipocytes. These results expand our understanding of catecholaminergic effects on BAT thermogenesis.

Computer design, synthesis, and bioactivity analyses of drugs like fingolimod used in the treatment of multiple sclerosis

Multiple sclerosis (MS) is a very common disease of vital importance. In the MS treatment, some drugs such as fingolimod which help to protect nerves from damage are used. The main goal of the drug therapy in MS is to take control of the inflammation which leads to the destruction of myelin and axons in nerve cell and thus prevent and stop the progression of the disease. Fingolimod (FTY720) is an orally active immunomodulatory drug that has been used for the treatment of relapsing-remitting multiple sclerosis. It is a sphingosine-1-phosphate receptor modulator which prevents lymphocytes from contributing to an autoimmune reaction by inhibiting egress of lymphocytes them from lymph nodes. In this study, we have computer designed, synthesized and characterized two novel derivatives of FTY720, F1-12h and F2-9, and have determined their underlying mechanism of their beneficial effect in SH-SY5Y, SK-N-SH, and U-118 MG cell lines. For this purpose, we first determined the regulation of the cAMP response element (CRE) activity and cAMP concentration by F1-12h and F2-9 together with FTY720 using pGL4.29 luciferase reporter assay and cAMP immunoassay, respectively. Then, we have determined their effect on MS- and GPCR-related gene expression profiles using custom arrays along with FTY720 treatment at non-toxic doses (EC10). It was found that both derivatives significantly activate CRE and increase cAMP concentration in all three cell lines, indicating that they activate cAMP pathway through cell surface receptors as FTY720 does. Furthermore, F1-12h and F2-9 modulate the expression of the pathway related genes that are important in inflammatory signaling, cAMP signaling pathway, cell migration as well as diverse receptor and transcription factors. Expression of the genes involved in myelination was also increased by the treatment with F1-12h and F2-9. In summary, our data demonstrate that the two novel FTY720 derivatives act as anti-inflammatory ultimately by influencing the gene expression via the cAMP and downstream transcription factor CRE pathway. In conclusion, F1-12h and F2-9 might contribute future therapies for autoimmune diseases such as multiple sclerosis.

Seizure Suppression by High Temperature via cAMP Modulation in Drosophila.

Bang-sensitive (BS) Drosophila mutants display characteristic seizure-like activity (SLA) and paralysis after mechanical shock . After high-frequency electrical stimulation (HFS) of the brain, they generate robust seizures at very low threshold voltage. Here we report an important phenomenon, which effectively suppresses SLA in BS mutants. High temperature causes seizure suppression in all BS mutants (parabss1, eas, sda) examined in this study. This effect is fully reversible and flies show complete recovery from BS paralysis once the temperature effect is nullified. High temperature induces an increase in seizure threshold after a brief pulse of heat shock (HS). By genetic screening, we identified the involvement of cAMP in the suppression of seizures by high temperature. We propose that HS induces adenylyl cyclase which in turn increases cAMP concentration which eventually suppresses seizures in mutant flies. In summary, we describe an unusual phenomenon, where high temperature can suppress SLA in flies by modulating cAMP concentration.

Effects of baicalin on alveolar fluid clearance and α-ENaC expression in rats with LPS-induced acute lung injury.

Baicalin has been reported to attenuate lung edema in the process of lung injury. However, the effect of baicalin on alveolar fluid clearance (AFC) and epithelial sodium channel (ENaC) expression has not been tested. Sprague-Dawley rats were anesthetized and intratracheally injected with either 1 mg/kg lipopolysaccharide (LPS) or saline vehicle. Baicalin with various concentrations (10, 50, and 100 mg/kg) was injected intraperitoneally 30 min before administration of LPS. Then lungs were isolated for measurement of AFC, cyclic adenosine monophosphate (cAMP) level, and cellular localization of α-ENaC. Moreover, mouse alveolar type II (ATII) epithelial cell line was incubated with baicalin (30 μmol/L), adenylate cyclase inhibitor SQ22536 (10 μmol/L), or cAMP-dependent protein kinase inhibitor (PKA) KT5720 (0.3 μmol/L) 15 min before LPS (1 μg/mL) incubation. Protein expression of α-ENaC was detected by Western blot. Baicalin increased cAMP concentration and AFC in a dose-dependent manner in rats with LPS-induced acute lung injury. The increase of AFC induced by baicalin was associated with an increase in the abundance of α-ENaC protein. SQ22536 and KT5720 prevented the increase of α-ENaC expression caused by baicalin in vitro. These findings suggest that baicalin prevents LPS-induced reduction of AFC by upregulating α-ENaC protein expression, which is activated by stimulating cAMP/PKA signaling pathway.

Influence of glycosaminoglycan from Mactra veneriformison antiplatelet aggregation

In this study, influence of glycosaminoglycan from Mactra veneriformis on antiplatelet aggregation in rats was studied. The results showed that glycosaminoglycan (GAG) from Mactra veneriformis could reduce the platele aggregation and the platelet adhesion rates of rats in vivo and in vitro (P&lt;0.05, P&lt;0.01), attenuate calcium ion concentration in rat platelets (P&lt;0.01), increase cAMP concentration in rat platelets (P&lt; 0.05, P&lt;0.01), diminish TXB2 concentrationin in rat plasma (P&lt;0.01), elevate 6-keto-PGF1a concentration in rat plasma (P&lt;0.05, P&lt;0.01) . This study suggests that GAG has obviously inhibitory effects on ADP-induced platelet aggregation in rats, which might account for its anti-thrombitic activity.

Pharmacological characterisation of the interaction between glycopyrronium bromide and indacaterol fumarate in human isolated bronchi, small airways and bronchial epithelial cells

BackgroundNowadays, there is a considerable gap in knowledge concerning the mechanism(s) by which long-acting β2-agonists (LABAs) and long-acting muscarinic antagonists (LAMAs) interact to induce bronchodilation. This study aimed to characterise the pharmacological interaction between glycopyrronium bromide and indacaterol fumarate and to identify the mechanism(s) leading to the bronchorelaxant effect of this interaction.MethodsThe effects of glycopyrronium plus indacaterol on the contractile tone of medium and small human isolated bronchi were evaluated, and acetylcholine and cAMP concentrations were quantified. The interaction was assessed by Bliss Independence approach.ResultsGlycopyrronium plus indacaterol synergistically inhibited the bronchial tone (medium bronchi, +32.51 % ± 7.86 %; small bronchi, +28.46 % ± 5.35 %; P < 0.05 vs. additive effect). The maximal effect was reached 140 min post-administration. A significant (P < 0.05) synergistic effect was observed during 9 h post-administration on the cholinergic tone, but not on the histaminergic contractility. Co-administration of glycopyrronium and indacaterol reduced the release of acetylcholine from the epithelium but not from bronchi, and enhanced cAMP levels in bronchi and epithelial cells (P < 0.05 vs. control), an effect that was inhibited by the selective KCa++ channel blocker iberiotoxin. The role of cAMP-dependent pathway was confirmed by the synergistic effect elicited by the adenylate cyclase activator forskolin on glycopyrronium (P < 0.05 vs. additive effect), but not on indacaterol (P > 0.05 vs. additive effect), with regard of the bronchial relaxant response and cAMP increase.ConclusionsGlycopyrronium/indacaterol co-administration leads to a synergistic improvement of bronchodilation by increasing cAMP concentrations in both airway smooth muscle and bronchial epithelium, and by decreasing acetylcholine release from the epithelium.

G-Protein-Coupled Receptor MrgD Is a Receptor for Angiotensin-(1-7) Involving Adenylyl Cyclase, cAMP, and Phosphokinase A.

Angiotensin (Ang)-(1-7) has cardiovascular protective effects and is the opponent of the often detrimental Ang II within the renin-angiotensin system. Although it is well accepted that the G-protein-coupled receptor Mas is a receptor for the heptapeptide, the lack in knowing initial signaling molecules stimulated by Ang-(1-7) prevented definitive characterization of ligand/receptor pharmacology as well as identification of further hypothesized receptors for the heptapeptide. The study aimed to identify a second messenger stimulated by Ang-(1-7) allowing confirmation as well as discovery of the heptapeptide's receptors. Ang-(1-7) elevates cAMP concentration in primary cells, such as endothelial or mesangial cells. Using cAMP as readout in receptor-transfected human embryonic kidney (HEK293) cells, we provided pharmacological proof that Mas is a functional receptor for Ang-(1-7). Moreover, we identified the G-protein-coupled receptor MrgD as a second receptor for Ang-(1-7). Consequently, the heptapeptide failed to increase cAMP concentration in primary mesangial cells with genetic deficiency in both Mas and MrgD Mice deficient in MrgD showed an impaired hemodynamic response after Ang-(1-7) administration. Furthermore, we excluded the Ang II type 2 receptor as a receptor for the heptapeptide but discovered that the Ang II type 2 blocker PD123319 can also block Mas and MrgD receptors. Our results lead to an expansion and partial revision of the renin-angiotensin system, by identifying a second receptor for Ang-(1-7), by excluding Ang II type 2 as a receptor for the heptapeptide, and by enforcing the revisit of such publications which concluded Ang II type 2 function by only using PD123319.

Effect of Caffeine on Cardiac Tissue Metabolism in Rabbits

Caffeine has been reported to influence skeletal muscle tissue metabolism at rest and during exercise (Goldstein et al 2010; Graham et al 1994). This study was designed to investigate the effect of caffeine on cardiac tissue metabolism in the rabbit.The study was carried out on adult male New Zealand rabbits (weighing 1.8–2.2Kg) divided into 3 groups (n=5). Group I rabbits were given 0.2ml/Kg of normal saline and served as control while groups II and III rabbits were administered with 2mg/Kg and 6mg/kg of caffeine respectively for 28 days. Blood samples (3.0ml/rabbit) were collected by retro orbital puncture to determine serum Myeloperoxidase (MPO) activity and nitrite concentration. The animals were sacrificed by cervical dislocation and cardiac tissue biopsies were collected on dry ice for biochemical and immunohistochemical analyses. Cardiac tissue glycogen concentration was determined with the aid of anthrone reagent. Cardiac tissue Carnitine Palmitol Transferase 1 (CPT1) activity and cAMP concentrations were determined by immunohistochemistry and colorimetry techniques respectively, with assay kits obtained from Biovision inc.The results showed that caffeine at 2mg/kg and 6mg/kg significantly inhibited MPO activity from 0.72±0.05 to 0.164±0.045 and 0.46±0.12 U/L respectively (p&lt;0.05). Caffeine at 2mg/kg had no significant effect on serum nitrite concentration but at 6mg/Kg, caffeine significantly increased nitrite concentration from 28.01±6.53 to 45.25±3.88μM (p&lt;0.05). Also caffeine at 2mg/kg and 6mg/kg increased cardiac tissue glycogen concentration from 15.62±0.73 to 40.69±6.35 and 38.82±6.91mg/100g, cAMP concentrations from 5.097±0.29 to 6.840±0.26 and 6.36±0.14 pmol/well and CPT1 activity from 18.3 to 20 and 25.2% respectively (p&lt;0.05).In conclusion, this study demonstrates that caffeine exhibits strong effects on cardiac tissue metabolism thereby causing cardioprotection, cardiac hyperemia, glycogen sparing, increased cAMP concentration and cardiac utilization of fatty acids as source of energy.