Cellular Cyclic AMP Levels Research Articles

Small Hepatitis B Virus Surface Antigen Promotes Hepatic Gluconeogenesis via Enhancing Glucagon/cAMP/Protein Kinase A/CREB Signaling.

Hepatitis B virus (HBV) is a major risk factor for serious liver diseases. The liver plays a unique role in controlling carbohydrate metabolism to maintain the glucose level within the normal range. Chronic HBV infection has been reported to associate with a high prevalence of diabetes. However, the detailed molecular mechanism underlying the potential association remains largely unknown. Here, we report that liver-targeted delivery of small HBV surface antigen (SHBs), the most abundant viral protein of HBV, could elevate blood glucose levels and impair glucose and insulin tolerance in mice by promoting hepatic gluconeogenesis. Hepatocytes with SHB expression also exhibited increased glucose production and expression of gluconeogenic genes glucose-6-phosphatase (G6pc) and phosphoenolpyruvate carboxykinase (PEPCK) in response to glucagon stimulation. Mechanistically, SHBs increased cellular levels of cyclic AMP (cAMP) and consequently activated protein kinase A (PKA) and its downstream effector cAMP-responsive element binding protein (CREB). SHBs-induced activation of CREB enhanced transcripts of gluconeogenic genes, thus promoting hepatic gluconeogenesis. The elevated cAMP level resulted from increased transcription activity and expression of adenylyl cyclase 1 (AC1) by SHBs through a binary E-box factor binding site (BEF). Taken together, we unveiled a novel pathogenic role and mechanism of SHBs in hepatic gluconeogenesis, and these results might highlight a potential target for preventive and therapeutic intervention in the development and progression of HBV-associated diabetes. IMPORTANCE Chronic HBV infection causes progressive liver damage and is found to be a risk factor for diabetes. However, the mechanism in the regulation of glucose metabolism by HBV remains to be established. In the current study, we demonstrate for the first time that the small hepatitis B virus surface antigen (SHBs) of HBV elevates AC1 transcription and expression to activate cAMP/PKA/CREB signaling and subsequently induces the expression of gluconeogenic genes and promotes hepatic gluconeogenesis both in vivo and in vitro. This study provides a direct link between HBV infection and diabetes and implicates that SHBs may represent a potential target for the treatment of HBV-induced metabolic disorders.

Nicotinamide Treatment Facilitates Mitochondrial Fission through Drp1 Activation Mediated by SIRT1-Induced Changes in Cellular Levels of cAMP and Ca2.

Mitochondrial autophagy (or mitophagy) is essential for mitochondrial quality control, which is critical for cellular and organismal health by attenuating reactive oxygen species generation and maintaining bioenergy homeostasis. Previously, we showed that mitophagy is activated in human cells through SIRT1 activation upon treatment of nicotinamide (NAM). Further, mitochondria are maintained as short fragments in the treated cells. In the current study, molecular pathways for NAM-induced mitochondrial fragmentation were sought. NAM treatment induced mitochondrial fission, at least in part by activating dynamin-1-like protein (Drp1), and this was through attenuation of the inhibitory phosphorylation at serine 637 (S637) of Drp1. This Drp1 hypo-phosphorylation was attributed to SIRT1-mediated activation of AMP-activated protein kinase (AMPK), which in turn induced a decrease in cellular levels of cyclic AMP (cAMP) and protein kinase A (PKA) activity, a kinase targeting S637 of Drp1. Furthermore, in NAM-treated cells, cytosolic Ca2+ was highly maintained; and, as a consequence, activity of calcineurin, a Drp1-dephosphorylating phosphatase, is expected to be elevated. These results suggest that NAD+-mediated SIRT1 activation facilitates mitochondrial fission through activation of Drp1 by suppressing its phosphorylation and accelerating its dephosphorylation. Additionally, it is suggested that there is a cycle of mitochondrial fragmentation and cytosolic Ca2+-mediated Drp1 dephosphorylation that may drive sustained mitochondrial fragmentation.

Melatonin ameliorates SGLT2 inhibitor-induced diabetic ketoacidosis by inhibiting lipolysis and hepatic ketogenesis in type 2 diabetic mice.

Sodium-glucose cotransporter-2 inhibitors (SGLT2i) are effective hypoglycemic agents that can induce glycosuria. However, there are increasing concerns that they might induce diabetic ketoacidosis. This study investigated the effect of melatonin on SGTL2i-induced ketoacidosis in insulin-deficient type 2 diabetic (T2D) mice. The SGLT2i dapagliflozin reduced blood glucose level and plasma insulin concentrations in T2D mice, but induced increases in the concentrations of plasma β-hydroxybutyrate, acetoacetate, and free fatty acid and a decrease in the concentration of plasma bicarbonate, resulting in ketoacidosis. Melatonin inhibited dapagliflozin-induced ketoacidosis without inducing any change in blood glucose level or plasma insulin concentration. In white adipose tissue, melatonin inhibited lipolysis and downregulated phosphorylation of PKA, HSL, and perilipin-1. In liver tissue, melatonin suppressed cellular cyclic AMP levels and downregulated phosphorylation of PKA, AMPK, and acetyl-CoA carboxylase (ACC). In addition, melatonin increased hepatic ACC activity, but decreased hepatic CPT1a activity and acetyl-CoA content. These effects of melatonin on lipolysis and hepatic ketogenesis were blocked by pretreatment with melatonin receptor antagonist or PKA activator. Collectively, these results suggest that melatonin can ameliorate SGLT2i-induced ketoacidosis by inhibiting lipolysis and hepatic ketogenesis though cyclic AMP/PKA signaling pathways in T2D mice. Thus, melatonin treatment may offer protection against SGLT2i-induced ketoacidosis.

A predictive computational model reveals that GIV/girdin serves as a tunable valve for EGFR-stimulated cyclic AMP signals.

Cellular levels of the versatile second messenger cyclic (c)AMP are regulated by the antagonistic actions of the canonical G protein → adenylyl cyclase pathway that is initiated by G-protein–coupled receptors (GPCRs) and attenuated by phosphodiesterases (PDEs). Dysregulated cAMP signaling drives many diseases; for example, its low levels facilitate numerous sinister properties of cancer cells. Recently, an alternative paradigm for cAMP signaling has emerged in which growth factor–receptor tyrosine kinases (RTKs; e.g., EGFR) access and modulate G proteins via a cytosolic guanine-nucleotide exchange modulator (GEM), GIV/girdin; dysregulation of this pathway is frequently encountered in cancers. In this study, we present a network-based compartmental model for the paradigm of GEM-facilitated cross-talk between RTKs and G proteins and how that impacts cellular cAMP. Our model predicts that cross-talk between GIV, Gαs, and Gαi proteins dampens ligand-stimulated cAMP dynamics. This prediction was experimentally verified by measuring cAMP levels in cells under different conditions. We further predict that the direct proportionality of cAMP concentration as a function of receptor number and the inverse proportionality of cAMP concentration as a function of PDE concentration are both altered by GIV levels. Taking these results together, our model reveals that GIV acts as a tunable control valve that regulates cAMP flux after growth factor stimulation. For a given stimulus, when GIV levels are high, cAMP levels are low, and vice versa. In doing so, GIV modulates cAMP via mechanisms distinct from the two most often targeted classes of cAMP modulators, GPCRs and PDEs.

Cyclic Nucleotide-Specific Phosphodiesterases as Potential Drug Targets for Anti-Leishmania Therapy.

The available treatments for leishmaniasis are less than optimal due to inadequate efficacy, toxic side effects, and the emergence of resistant strains, clearly endorsing the urgent need for discovery and development of novel drug candidates. Ideally, these should act via an alternative mechanism of action to avoid cross-resistance with the current drugs. As cyclic nucleotide-specific phosphodiesterases (PDEs) of Leishmania major have been postulated as putative drug targets, a series of potential inhibitors of Leishmania PDEs were explored. Several displayed potent and selective in vitro activity against L. infantum intracellular amastigotes. One imidazole derivative, compound 35, was shown to reduce the parasite loads in vivo and to increase the cellular cyclic AMP (cAMP) level at in a dose-dependent manner at just 2× and 5× the 50% inhibitory concentration (IC50), indicating a correlation between antileishmanial activity and increased cellular cAMP levels. Docking studies and molecular dynamics simulations pointed to imidazole 35 exerting its activity through PDE inhibition. This study establishes for the first time that inhibition of cAMP PDEs can potentially be exploited for new antileishmanial chemotherapy.

Gastrin Induces Nuclear Export and Proteasome Degradation of Menin in Enteric Glial Cells

The multiple endocrine neoplasia, type 1 (MEN1) locus encodes the nuclear protein and tumor suppressor menin. MEN1 mutations frequently cause neuroendocrine tumors such as gastrinomas, characterized by their predominant duodenal location and local metastasis at time of diagnosis. Diffuse gastrin cell hyperplasia precedes the appearance of MEN1 gastrinomas, which develop within submucosal Brunner's glands. We investigated how menin regulates expression of the gastrin gene and induces generation of submucosal gastrin-expressing cell hyperplasia. Primary enteric glial cultures were generated from the VillinCre:Men1FL/FL:Sst-/- mice or C57BL/6 mice (controls), with or without inhibition of gastric acid by omeprazole. Primary enteric glial cells from C57BL/6 mice were incubated with gastrin and separated into nuclear and cytoplasmic fractions. Cells were incubated with forskolin and H89 to activate or inhibit protein kinase A (a family of enzymes whose activity depends on cellular levels of cyclic AMP). Gastrin was measured in blood, tissue, and cell cultures using an ELISA. Immunoprecipitation with menin or ubiquitin was used to demonstrate post-translational modification of menin. Primary glial cells were incubated with leptomycin b and MG132 to block nuclear export and proteasome activity, respectively. We obtained human duodenal, lymph node, and pancreatic gastrinoma samples, collected from patients who underwent surgery from 1996 through 2007 in the United States or the United Kingdom. Enteric glial cells that stained positive for glial fibrillary acidic protein (GFAP+) expressed gastrin de novo through a mechanism that required PKA. Gastrin-induced nuclear export of menin via cholecystokinin B receptor (CCKBR)-mediated activation of PKA. Once exported from the nucleus, menin was ubiquitinated and degraded by the proteasome. GFAP and other markers of enteric glial cells (eg, p75 and S100B), colocalized with gastrin in human duodenal gastrinomas. MEN1-associated gastrinomas, which develop in the submucosa, might arise fromenteric glial cells through hormone-dependent PKA signaling. This pathway disrupts nuclear menin function, leading to hypergastrinemia and associated sequelae.

GPCRomic Analysis of Human Lung Fibroblasts

Many interstitial lung diseases are associated with increased numbers of fibroblast/myofibroblasts and increased deposition of extracellular matrix (ECM) proteins. Such fibroproliferative responses can lead to pulmonary fibrosis, a disease setting with very limited treatment options. Previous studies have shown that the conversion of fibroblasts into myofibroblasts and excessive deposition of ECM proteins can be regulated by the cellular level of cyclic AMP (cAMP), the synthesis of which is regulated by G‐protein signaling and G protein‐coupled receptors (GPCRs). We thus tested the hypothesis that expression of GPCRs (in particular, previously unrecognized GPCRs) by lung fibroblasts may identify novel regulators of the function of lung fibroblasts and potentially new therapeutic targets for lung fibrosis. We used real‐time PCR GPCR arrays to identify and quantify non‐chemosensory GPCRs expressed in lung fibroblasts and detected on average ~ 100 such commonly expressed receptors in low passage cultures of multiple primary isolates of human lung fibroblasts. To ascertain if the GPCR expression profile changes during the fibroblast to myofibroblast conversion, we treated fibroblasts in vitro with TGFβ1 (10 ng/ml, 48 hr). TGFβ1 treatment prominently increased the expression of pro‐fibrotic markers (e.g., collagens Iα1, Iα2, III; α‐smooth muscle actin; plasminogen activator inhibitor‐1), thus mimicking what occurs in interstitial lung diseases. GPCRomic analysis identified multiple GPCRs that were uniquely expressed in response to TGFβ1. Lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) also revealed the selective expression of a sub‐set of GPCRs. However, TGFβ1 treatment of control fibroblasts does not fully mimic the changes in GPCR expression of fibroblasts from patients with IPF. We conclude that GPCRomic analysis identifies GPCRs associated with (and that may contribute to) the fibrotic state in the lung and thus, may be novel therapeutic targets for lung fibrosis. In addition, our results imply that TGFβ1 is likely not the sole determinant of changes in GPCR expression that occur in the fibrotic state.Support or Funding InformationSupported by Bristol Myers Squibb and NIH T32 HL 098062‐04

T-2 toxin regulates steroid hormone secretion of rat ovarian granulosa cells through cAMP-PKA pathway

T-2 toxin is a secondary metabolite produced by Fusarium genus and is a common contaminant in food and feedstuffs of cereal origin. In porcine granulosa cells(GC), T-2 toxin has been shown to inhibit the steroidogenesis; however, the mechanism has not been well understood. Gonadotropin-stimulated steroidogenesis is regulated by the cAMP-PKA pathway. In this study, we investigated potential mechanisms for T-2 toxin-induced reproductive toxicity focusing on the critical steps of the cAMP-PKA pathway affected by T-2 toxin. We first analyzed the effects of T-2 toxin on progesterone and estrogen production in rat granulosa cells. For this purpose the granulosa cells were cultured for 48h in 10% fetal bovine serum-containing medium followed by 24h in serum-free medium containing FSH (10ng/ml) and androstenedione (3ng/ml), both are required for normal steroidogenesis. Treatment of these cells with T-2 toxin dose-dependently inhibited the growth of cells and the steroid hormone production. Cellular cyclic AMP levels were dose-dependently inhibited by T-2 toxin (0, 1, 10 and 100nM, 24h). Furthermore, we found that although the induction of progesterone by 8-Br-cAMP (a FSH mimetic) and 22R-HC (substrate for progesterone) could both be inhibited by T-2 toxin treatment, the T-2-imposed inhibitory effects could be reversed by increasing doses of 22R-HC, while increasing 8-Br-cAMP had no effects, suggesting that T2 toxin targeted at distinct mechanisms. cAMP-stimulated steroidogenic acute regulatory protein (StAR) is a rate limiting protein in progesterone synthesis. Exposure to T2 toxin caused significant suppression of StAR expression as determined by Western blotting and semi-quantitative RT-PCR suggesting StAR is a sensitive target for T-2 toxin. Taken together, our results strongly suggest that T2 toxin inhibits steroidogenesis by suppressing cAMP-PKA pathway and StAR is a target for T-2-toxin. The antisteroidogenesis effects were observable at low T-2 dose (1ng/ml) suggesting T-2 toxin has an endocrine disruptive effect.

Cyclic AMP reverses the pro‐fibrogenic phenotype of cardiac myofibroblasts (850.3)

Previous data have shown that increased cellular levels of cyclic AMP (cAMP) can prevent the transformation of cardiac fibroblast (CFs) into pro‐fibrogenic myofibroblasts (MyoFbs), a transformation that precedes increased extracellular matrix deposition and cardiac fibrosis. We have now investigated two additional roles for cAMP: 1) contribution to the MyoFb phenotype and 2) reversal of the MyoFb phenotype. We found that MyoFbs have lower cAMP content than do CFs, and in addition, have decreased mRNA and protein expression of isoforms of adenylyl cyclase (AC), including AC5 and AC6, the two major AC isoforms, and increased expression of several phosphodiesterase isoforms. Incubation of primary isolates of adult rat CFs with a pro‐fibrotic stimulus, TGFβ1 (10ng/mL, 48hr), prominently increases mRNA and protein expression of collagen Iα1, Iα2, III, α‐smooth muscle actin, plasminogen activator inhibitor‐1 and cellular contractile state. All these hallmarks of the fibrogenic state were reversed in MyoFbs incubated with 10µM forskolin (a direct AC activator). Furthermore, incubation with N6‐Phe‐cAMP (a PKA selective analog) also reversed the pro‐fibrogenic state. We conclude that MyoFbs have a lower level of cAMP and that increasing cAMP content can reverse their pro‐fibrotic state. Approaches to increase cAMP in MyoFbs may provide a means to treat cardiac fibrosis.Grant Funding Source: NIH

Regulation of the inflammatory response of vascular endothelial cells by EPAC1

Life-threatening diseases of the cardiovascular system, like atherosclerosis, are exacerbated by unwanted inflammation within the structures of large blood vessels. This inflammation involves increased permeability of the vascular endothelial cells (VECs) that form the lining of blood vessels, leading to exaggerated extravasation of blood components and accumulation of fluid in the extravascular space. This results in tissue dysfunction and increased secretion of chemokines that attract leukocytes and monocytes to the inflamed endothelium. Cyclic AMP is synthesized in VECs in response to endogenous Gs-coupled receptors and is known to limit cytokine action and reduce endothelial hyperpermeability induced by multiple pro-inflammatory stimuli. The mechanisms underlying this anti-inflammatory action of cyclic AMP are now being elucidated and it is becoming clear that the cyclic AMP sensor, exchange protein activated by cyclic AMP (EPAC1), appears to play a key role in suppressing unwanted inflammation. EPAC1 mediates at least three anti-inflammatory pathways in VECs by down-regulating inflammatory signalling through the induction of the suppressors of cytokine signalling 3 (SOCS-3) gene, limiting integrin-dependent vascular permeability and enhancing endothelial barrier function through the stabilization of VE-cadherin junctions. Given that manipulation of cellular cyclic AMP levels currently forms the basis of many effective pharmaceuticals and that EPAC1 is involved in multiple anti-inflammatory protective processes in VECs, does this make EPAC1 an attractive target for the development of activators capable of eliciting a coordinated programme of 'protection' against the development of endothelial dysfunction? Here we discuss whether EPAC1 represents an attractive therapeutic target for limiting endothelial dysfunction associated with cardiovascular diseases like atherosclerosis. LINKED ARTICLES This article is part of a themed section on Novel cAMP Signalling Paradigms. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.166.issue-2.

The signaling mechanisms mediating the inhibitory effect of TCH-1116 on formyl peptide-stimulated superoxide anion generation in neutrophils

In fMLP (formyl-Met-Leu-Phe)-stimulated rat neutrophils, a mixture of regioisomers benzo[a]furo[2,3-c]phenazine-10-carboxylic acid and benzo[a]furo[2,3-c]phenazine-11-carboxylic acid (TCH-1116) inhibited O2- (superoxide anion) generation, which was not mediated by scavenging the generated O2- or by a cytotoxic effect on neutrophils. TCH-1116 had no effect on the arachidonic acid-induced NADPH oxidase activation in a cell-free system, whereas it effectively attenuated the phosphorylation of Ser residues in p47phox and the association between p47phox and p22phox in fMLP-stimulated neutrophils. The interaction of p47phox with PKC (protein kinase C) isoforms (α, βI, βII, δ and ζ) was attenuated by TCH-1116, whereas TCH-1116 did not affect the PKC isoforms membrane translocation, phosphorylation (Ser660) and kinase activity. TCH-1116 effectively attenuated the association between PKB/Akt (protein kinase B) and p47phox, Akt phosphorylation (Thr308/Ser473) and kinase activities of Akt and human recombinant PDK (3-phosphoinositide-dependent kinase) 1, whereas it had no effect on recruitment of Akt, phospho-PDK1 (Ser241) and p110γ to membrane. Moreover, the interaction of p21-activated kinase (PAK) 1 with p47phox and the phosphorylation of PAK1 (Thr423 but not Ser144) were inhibited by TCH-1116, but without affecting the membrane recruitment of PAK1. The cellular cyclic AMP level was not changed by TCH-1116. Taken together, these results suggest that TCH-1116 inhibits fMLP-stimulated O2- generation in rat neutrophils through the blockade of PKC, Akt and PAK signaling pathways.

During autophagy mitochondria elongate, are spared from degradation and sustain cell viability

SummaryA plethora of cellular processes, including apoptosis, depend on regulated changes in mitochondrial shape and ultrastructure. Scarce is our understanding of the role of mitochondria and of their morphology during autophagy, a bulk degradation and recycling process of eukaryotic cells’ constituents. Here we show that mitochondrial morphology determines the cellular response to macroautophagy. When autophagy is triggered, mitochondria elongate in vitro and in vivo. Upon starvation cellular cAMP levels increase and protein kinase A (PKA) becomes activated. PKA in turn phosphorylates the pro-fission dynamin related protein 1 (DRP1) that is therefore retained in the cytoplasm, leading to unopposed mitochondrial fusion. Elongated mitochondria are spared from autophagic degradation, possess more cristae, increase dimerization and activity of ATP synthase, and maintain ATP production. When elongation is genetically or pharmacologically blocked, mitochondria conversely consume ATP, precipitating starvation-induced death. Thus, regulated changes in mitochondrial morphology determine the fate of the cell during autophagy.

Expression and function of cannabinoid receptors in mouse islets

The endocannabinoid system plays a key role in energy homeostasis, with agonists and antagonists of CB1 receptors acting centrally to stimulate and inhibit food intake, respectively. In addition to their established effects on the central nervous system, cannabinoid receptor agonists also exert peripheral effects by modulating cellular cyclic AMP and calcium levels and there have been reports that they regulate β-cell function. However, the few reports to date on islet expression of cannabinoid receptors and effects of agonists on insulin secretion have failed to reach a consensus. We have therefore investigated cannabinoid receptor expression by mouse islet β-and α-cells and the effects of selective receptor agonists on cyclic AMP and calcium levels, and on dynamic insulin secretory responses. CB1 and CB2 mRNA and protein expression by islets was detected by RT-PCR and western blotting respectively, and cellular location of the receptors was identified by immunohistochemistry with insulin and glucagon antibody co-staining. Cyclic AMP generation was quantified by enzyme immunoassay and changes in calcium levels were measured by microfluorimetry of Fura-2-loaded mouse islet cells. Dynamic insulin secretion was quantified by radioimmunoassay after perifusion of isolated islets. We found that mouse islets expressed both CB1 and CB2 receptors, and they were localised to β-cells. Activation of mouse β-cell CB1 and CB2 receptors resulted in decreased cyclic AMP, increased calcium and potentiation of glucose-stimulated insulin secretion. Thus, activation of islet cannabinoid receptors by locally produced endocannabinoids such as 2-aminoglycerol may be another regulatory pathway by which islets stimulate insulin secretion to maintain glucose homeostasis.

Thyroglobulin (Tg) induces thyroid cell growth in a concentration-specific manner by a mechanism other than thyrotropin/cAMP stimulation

Thyroglobulin (Tg), a major product of the thyroid gland, serves as a macromolecular precursor of thyroid hormone biosynthesis. In addition, Tg stored in the thyroid follicles is a potent regulator of thyroid-specific gene expression. In conjunction with thyroid stimulating hormone (TSH) and iodide, Tg regulates thyroid follicle function, which is the minimal functional unit of the thyroid gland. In the present study, we show that Tg stimulates growth of FRTL-5 thyroid cells in the absence of TSH, insulin and serum. Unlike TSH, Tg did not increase cellular cyclic AMP (cAMP) levels; rather, the TSH signal counteracted Tg-induced cell growth. A specific inhibitor of A-kinase, H-89, did not modulate the effect of Tg. Tg increased kinase activity of Akt to the same level as TSH, insulin and 5% serum, while LY294002 abolished Tg-induced growth. Interestingly, low Tg concentrations maximized growth-promotion activity and induction of the apical iodide transporter (PDS; SLC26A4), whereas high Tg concentrations suppressed both cell growth and the expression of thyroid-specific genes. These results suggest that a low levels of Tg in the follicular lumen might stimulates cell growth and iodide transport to accelerate the iodide organification process; however, elevated Tg levels in the follicle might then shut down all of these functions.

Anthrax Edema Toxin Induces Maturation of Dendritic Cells and Enhances Chemotaxis towards Macrophage Inflammatory Protein 3β

Bacillus anthracis secretes two bipartite toxins, edema toxin (ET) and lethal toxin (LT), which impair immune responses and contribute directly to the pathology associated with the disease anthrax. Edema factor, the catalytic subunit of ET, is an adenylate cyclase that impairs host defenses by raising cellular cyclic AMP (cAMP) levels. Synthetic cAMP analogues and compounds that raise intracellular cAMP levels lead to phenotypic and functional changes in dendritic cells (DCs). Here, we demonstrate that ET induces a maturation state in human monocyte-derived DCs (MDDCs) similar to that induced by lipopolysaccharide (LPS). ET treatment results in downregulation of DC-SIGN, a marker of immature DCs, and upregulation of DC maturation markers CD83 and CD86. Maturation of DCs by ET is accompanied by an increased ability to migrate toward the lymph node-homing chemokine macrophage inflammatory protein 3beta, like LPS-matured DCs. Interestingly, cotreating with LT differentially affects the ET-induced maturation of MDDCs while not inhibiting ET-induced migration. These findings reveal a mechanism by which ET impairs normal innate immune function and may explain the reported adjuvant effect of ET.

Inhibition of phosphodiesterase has an additive effect on estrogen's ability to inhibit collagen synthesis in vascular smooth muscle cells

Several studies have shown that estrogen has the ability to decrease collagen synthetic rates in vascular smooth muscle cells (VSMCs) by increasing cellular cyclic AMP (cAMP) levels. Phosphodiesterase inhibitors have also been shown to inhibit collagen synthesis in VSMCs presumably by preventing the degradation of cAMP. Since estrogens and phosphodiesterase inhibitors are used clinically, it is important to determine the potential for phosphodiesterase inhibitors to potentiate estrogen's ability to inhibit collagen synthesis in VSMCs. The results of the present study demonstrate that the phosphodiesterase inhibitors cilostamide and Ro-20-1724 had an additive effect on estrogen's ability to inhibit collagen synthesis in VSMC. Also, the data suggests that phosphodiesterase inhibitors mediated this additive effect by increasing cellular levels of cAMP.

Mutation at Different Sites in the Nostoc punctiforme cyaC Gene, Encoding the Multiple-Domain Enzyme Adenylate Cyclase, Results in Different Levels of Infection of the Host Plant Blasia pusilla

The filamentous cyanobacterium Nostoc punctiforme forms symbioses with plants. Disruption of the catalytic domain of the N. punctiforme adenylate cyclase (CyaC) significantly increased symbiotic competence, whereas reduced infectivity was observed in a mutant with a disruption close to the N terminus of CyaC. The total cellular cyclic AMP levels were significantly reduced in both mutants.

Effect of capsaicin on plasma and tissue levels of insulin-like growth factor-I in spontaneously hypertensive rats

Objective Plasma levels of insulin-like growth factor-I (IGF-I), an important substance for maintaining cardiovascular homeostasis, are lower in spontaneously hypertensive rats (SHR) than in normotensive Wistar Kyoto rats (WKY). Calcitonin gene-related peptide (CGRP) increases IGF-I production in vitro and in vivo, suggesting that stimulation of sensory neurons might increase the production of IGF-I in SHR. Design Levels of CGRP and IGF-I in plasma, kidneys and heart in WKY and SHR and cellular cyclic AMP levels in dorsal root ganglion neurons (DRGs) isolated from WKY and SHR were measured by an ELISA-based method. Results Levels of CGRP and IGF-I in plasma, kidneys and heart of SHR were about half of those of WKY. Administration of capsaicin significantly increased levels of CGRP and IGF-I in plasma and tissues of SHR to the levels in WKY and these increases were completely reversed by pretreatment with capsazepine, an inhibitor of vanilloid receptor-1 activation. CGRP release and cellular levels of cAMP in DRGs isolated from SHR were significantly lower than those in DRGs isolated from WKY. Capsaicin increased both CGRP release and cellular cAMP levels in DRGs isolated from SHR to the levels in DRGs isolated from WKY. Conclusions Sensory neuron activation might be lower in SHR than in WKY probably due to decreased production of cAMP in sensory neurons, explaining why IGF-I levels in plasma and tissues are lower in SHR than in WKY.

Mechanisms of action of VIP and PACAP in the stimulation of insulin release.

VIP and PACAP stimulate insulin release by interaction with the VIP-2/PACAP-3 receptor on the beta cell. Activation of the receptor results in Gs-mediated stimulation of adenylyl cyclase and increased cellular cyclic AMP levels. Increased cyclic AMP results in a small and transient increase in [Ca2+]i, which is likely to have only a small and transient effect on the secretion rate. Cyclic AMP also potentiates insulin secretion by an as yet unknown action at a distal site. A third action of VIP and PACAP is responsible for the continued stimulation of insulin secretion after the levels of cyclic AMP and [Ca2+]i have returned to basal values. This third pathway, which is identified at present only by its sensitivity to low concentrations of wortmannin, plays a major role in the prolonged stimulation of insulin release by VIP and PACAP.

Inducing coproporphyria in rat hepatocyte cultures using cyclic AMP and cyclic AMP-releasing agents

Cyclic AMP (c-AMP), added on its own to rat hepatocyte cultures, caused a marked accumulation of coproporphyrin III. The results obtained by comparing the effect of c-AMP to that of exogenous 5-aminolevulinate (ALA), and from adding c-AMP and ALA together, indicated that the coproporphyrinogen III metabolism was blocked, even though no inhibition of the relevant enzyme, coproporphyrinogen oxidase, could be demonstrated. Preferential accumulation of coproporphyrin could also be produced in cultures of rat hepatocytes by agents that raise the cellular levels of cyclic AMP, such as glucagon. The effect of supplementing the culture medium with triiodothyronine (T3) on the response of rat hepatocytes to c-AMP was also investigated. T3, which is known to stimulate mitochondrial respiration, uncoupling O2 consumption from ATP synthesis, produced a c-AMP-like effect when given on its own and potentiated the effect of c-AMP, with an apparent increase in the severity of the metabolic block. It is suggested that an oxidative mechanism may be activated in c-AMP and T3-induced coproporphyria, preferentially involving the mitochondrial compartment, leading to oxidation of porphyrinogen intermediates of haem biosynthesis, especially coproporphyrinogen. Coproporphyin, the fully oxidized aromatic derivative produced, cannot be metabolized and will therefore accumulate.