Role Of cAMP Research Articles

CAMP promotes differentiation of rodent neuronal progenitor cells

Numerous studies have described neuronal differentiation of neural progenitor cells derived from fetal tissue in vitro, but the biochemical mechanisms underlying this process remain largely unknown. In the present study, the role of cAMP in promoting functional maturation of neuronal progenitor cells (NPCs) from the subventricular zone (SVZ) of rodent fetal brain was investigated. NPCs were extracted from telencephalic vesicles of E14 rat embryos and then expanded in medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). A mature neuronal fate was induced by: i) withdrawal of growth factors (basal condition); ii) addition of brain-derived neurotrophic factor (BDNF); or iii) addition of isobutylmethylxantine (IBMX). Whole cell patch clamping assessed electrophysiological properties. Immunocyto - chemistry for MAP2 confirmed neuronal differentiation. Quantification of neuronal cells, and determination of their electrophysiological properties, was performed in the cited experimental groups. IBMX significantly enhanced the yield of MAP2-positive neurons in the culture system (3.7-fold increase). Application of a one-week differentiation protocol under IBMX induced functional maturity. MAP2-positive cells presented large Na- and K- voltagedependent currents, fired bursts of action potentials, and exhibited spontaneous synaptic activity. Further, IBMX proved more effective than BDNF for promoting neuronal maturation, leading to higher evoked peak currents and current densities, as well as greater yields of cells firing action potentials and presenting active synaptic contacts. Our data indicates the importance of cAMP-dependent mechanisms for maturation of neuronal progenitor cells in vitro. This knowledge can enable future manipulation of neurogenesis in vivo in order to promote the intrinsic regenerative capacity of the central nervous system.

CAMP-secretion coupling is impaired in diabetic GK/Par rat β-cells: a defect counteracted by GLP-1

cAMP-raising agents with glucagon-like peptide-1 (GLP-1) as the first in class, exhibit multiple actions that are beneficial for the treatment of type 2 diabetic (T2D) patients, including improvement of glucose-induced insulin secretion (GIIS). To gain additional insight into the role of cAMP in the disturbed stimulus-secretion coupling within the diabetic β-cell, we examined more thoroughly the relationship between changes in islet cAMP concentration and insulin release in the GK/Par rat model of T2D. Basal cAMP content in GK/Par islets was significantly higher, whereas their basal insulin release was not significantly different from that of Wistar (W) islets. Even in the presence of IBMX or GLP-1, their insulin release did not significantly change despite further enhanced cAMP accumulation in both cases. The high basal cAMP level most likely reflects an increased cAMP generation in GK/Par compared with W islets since 1) forskolin dose-dependently induced an exaggerated cAMP accumulation; 2) adenylyl cyclase (AC)2, AC3, and G(s)α proteins were overexpressed; 3) IBMX-activated cAMP accumulation was less efficient and PDE-3B and PDE-1C mRNA were decreased. Moreover, the GK/Par insulin release apparatus appears less sensitive to cAMP, since GK/Par islets released less insulin at submaximal cAMP levels and required five times more cAMP to reach a maximal secretion rate no longer different from W. GLP-1 was able to reactivate GK/Par insulin secretion so that GIIS became indistinguishable from that of W. The exaggerated cAMP production is instrumental, since GLP-1-induced GIIS reactivation was lost in the presence the AC blocker 2',5'-dideoxyadenosine. This GLP-1 effect takes place in the absence of any improvement of the [Ca(2+)](i) response and correlates with activation of the cAMP-dependent PKA-dependent pathway.

Evolution of developmental cyclic adenosine monophosphate signaling in the Dictyostelia from an amoebozoan stress response.

The Dictyostelid social amoebas represent one of nature’s several inventions of multicellularity. Though normally feeding as single cells, nutrient stress triggers the collection of amoebas into colonies that form delicately shaped fruiting structures in which the cells differentiate into spores and up to three cell types to support the spore mass. Cyclic adenosine monophosphate (cAMP) plays a very dominant role in controlling morphogenesis and cell differentiation in the model species Dictyostelium discoideum. As a secreted chemoattractant cAMP coordinates cell movement during aggregation and fruiting body morphogenesis. Secreted cAMP also controls gene expression at different developmental stages, while intracellular cAMP is extensively used to transduce the effect of other stimuli that control the developmental program. In this review, I present an overview of the different roles of cAMP in the model D. discoideum and I summarize studies aimed to resolve how these roles emerged during Dictyostelid evolution.

Cyclic AMP signalling in mycobacteria: redirecting the conversation with a common currency

cAMP is an ancient second messenger, and is used by many organisms to regulate a wide range of cellular functions. Mycobacterium tuberculosis complex bacteria are exceptional in that they have genes for at least 15 biochemically distinct adenylyl cyclases, the enzymes that generate cAMP. cAMP-associated gene regulation within tubercle bacilli is required for their virulence, and secretion of cAMP produced by M. tuberculosis bacteria into host macrophages disrupts the host's immune response to infection. In this review, we discuss recent advances in our understanding of the means by which cAMP levels are controlled within mycobacteria, the importance of cAMP to M. tuberculosis during host infection, and the role of cAMP in mycobacterial gene regulation. Understanding the myriad aspects of cAMP signalling in tubercle bacilli will establish new paradigms for cAMP signalling, and may contribute to new approaches for prevention and/or treatment of tuberculosis disease.

Fitting MITF regulation of PDE4D3 into the skin pigmentation puzzle

Fitting MITF regulation of PDE4D3 into the skin pigmentation puzzle

CXCL8 attenuates chemoattractant-induced equine neutrophil migration

The chemokine, CXCL8, is a potent chemoattractant but it has also been shown to attenuate the migratory response of human neutrophils to the bacterial peptide, FMLP; this could lead to retention of cells in infected tissue and, potentially, to enhanced clearance of bacteria. This study has examined the effect of CXCL8 on equine neutrophil migration and adherence in response to PAF and LTB 4, chemoattractants that may play a role in non-infectious inflammatory conditions of the horse associated with neutrophil recruitment to the target tissue. The effects of CXCL8 on PAF- and LTB 4-induced responses were determined using a ChemoTx plate migration assay and by measuring adhesion to protein-coated plastic. The CXCR1/2 antagonist, SB225002, was used to investigate whether the observed effects were receptor mediated and the role of cAMP was examined by measuring intracellular cAMP following exposure to agonists alone and in combination and by establishing the effect of dibutyryl cAMP on neutrophil migration. CXCL8, LTB 4 and PAF each induced migration and adhesion. Exposure of neutrophils to a combination of CXCL8 and PAF reduced the magnitude of the responses to that of unstimulated cells. In contrast, although the effect was less than additive, the response to co-stimulation with CXCL8 and LTB 4 were not nearly as pronounced. CXCL8 acted in a receptor mediated manner, the attenuation of PAF-induced responses being reversed by SB225002 at a concentration that blocks CXCR2. CXCL8, PAF and LTB 4 alone increased intracellular cAMP. In co-incubation studies, combination of CXCL8 with PAF led to an additive increase in cAMP whereas no increase above that obtained in response to LTB 4 alone was seen. Dibutyryl cAMP significantly reduced neutrophil migration in response to either CXCL8 or PAF alone. These results demonstrate that CXCL8, in addition to being a potent chemoattractant and pro-adhesive molecule for equine neutrophils, is able to attenuate responses to PAF and, to a much lesser extent, LTB 4. This effect, which appears to be CXCR2-mediated and cAMP dependent, could lead in vivo to trapping of cells at sites of inflammation resulting potentially in either enhanced clearance of injurious stimuli or increased local tissue damage by activated cells.

The Role of the Concentration Camps in the Nazi Repression of Prostitutes, 1933-9

This article uses prostitutes as a case study in order to investigate the role of the early concentration camps as centres of detention for social deviants. In contrasting the intensification of repressive policies towards prostitutes against narratives which demonstrate the unexpectedly lax treatment of these women, it explores what the reasons behind these contradictions might have been, and what this demonstrates about the development of these institutions. It asks the following questions. How and why were prostitutes interned? Which bureaucrats were responsible for incarcerating these women and what did they view the role of the camp to be? Were such policies centrally directed or the product of local decision-making? Through asking these questions, the article explores to what extent these camps were unique as mechanisms for the repression and marginalization of prostitutes.

CAMP Mediators of Pulsatile Insulin Secretion from Glucose-stimulated Single β-Cells

Pulsatile insulin release from glucose-stimulated beta-cells is driven by oscillations of the Ca(2+) and cAMP concentrations in the subplasma membrane space ([Ca(2+)](pm) and [cAMP](pm)). To clarify mechanisms by which cAMP regulates insulin secretion, we performed parallel evanescent wave fluorescence imaging of [cAMP](pm), [Ca(2+)](pm), and phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) in the plasma membrane. This lipid is formed by autocrine insulin receptor activation and was used to monitor insulin release kinetics from single MIN6 beta-cells. Elevation of the glucose concentration from 3 to 11 mm induced, after a 2.7-min delay, coordinated oscillations of [Ca(2+)](pm), [cAMP](pm), and PIP(3). Inhibitors of protein kinase A (PKA) markedly diminished the PIP(3) response when applied before glucose stimulation, but did not affect already manifested PIP(3) oscillations. The reduced PIP(3) response could be attributed to accelerated depolarization causing early rise of [Ca(2+)](pm) that preceded the elevation of [cAMP](pm). However, the amplitude of the PIP(3) response after PKA inhibition was restored by a specific agonist to the cAMP-dependent guanine nucleotide exchange factor Epac. Suppression of cAMP formation with adenylyl cyclase inhibitors reduced already established PIP(3) oscillations in glucose-stimulated cells, and this effect was almost completely counteracted by the Epac agonist. In cells treated with small interfering RNA targeting Epac2, the amplitudes of the glucose-induced PIP(3) oscillations were reduced, and the Epac agonist was without effect. The data indicate that temporal coordination of the triggering [Ca(2+)](pm) and amplifying [cAMP](pm) signals is important for glucose-induced pulsatile insulin release. Although both PKA and Epac2 partake in initiating insulin secretion, the cAMP dependence of established pulsatility is mediated by Epac2.

Involvement of cAMP in nerve growth factor-triggered p35/Cdk5 activation and differentiation in PC12 cells

The signaling mechanisms underlying cell differentiation have been extensively studied with the use of rat PC12 cells as a model system. Nerve growth factor (NGF) is a trophic factor inducing PC12 cell differentiation through the activation of the p35/cyclin-dependent kinase 5 (Cdk5) complex. It has been reported that adenylyl cyclase activation and cAMP production may be involved in NGF-dependent actions. Our previous results indicate that cAMP activates the p35/Cdk5 complex in reproductive cells. Therefore, the role of cAMP in NGF-triggered p35/Cdk5 activation and PC12 differentiation was interesting to explore. Our results indicate that roscovitine, a molecular inhibitor of Cdk5, blocks cAMP-triggered PC12 differentiation, which was evaluated by neurite initiation, a decrease in proliferation, and cell cycle G(1) arrest. The following data show that cAMP treatment increased Cdk5 activity through p35 upregulation. cAMP downstream components, protein kinase A (PKA) and phosphorylated cAMP response element binding protein (CREB), are involved in this regulation. The immunocytochemical results indicate that PKA inhibition disrupted cAMP-triggered p35/Cdk5 localization in PC12 cells. In addition, adenylyl cyclase inhibition was found to diminish NGF-induced intracellular cAMP production, CREB phosphorylation, and p35 expression. The cAMP antagonist and the PKA inhibitors reduced NGF-induced p35 expression. Finally, NGF-triggered PC12 differentiation was partially decreased by adenylyl cyclase or PKA inhibitors. In conclusion, these results demonstrate that cAMP may play a role in NGF-p35/Cdk5-dependent PC12 differentiation.

Elucidation of Mechanisms of the Reciprocal Cross Talk between Gonadotropin-Releasing Hormone and Prostaglandin Receptors

We recently described a novel GnRH receptor signaling pathway mediated by the prostaglandins (PGs) F(2alpha) and PGI(2), which acts through an autocrine/paracrine modality to limit autoregulation of the GnRH receptor and inhibit LH but not FSH release. Here we further explore the cross talk between GnRH and the PG receptors. GnRH stimulates arachidonic acid (AA) release from LbetaT2 gonadotrope cells via the Ca(2+)-independent phospholipase A(2) (iPLA(2)) and not via the more common Ca(2+)-dependent cytosolic phospholipase A(2)alpha (cPLA(2)alpha). AA release was followed by a marked induction of cyclooxygenase (COX)-1 and COX-2 by GnRH via the protein kinase C/c-Src/phosphatidylinositol 3-kinase/MAPK pathway. COX-2 transcription by GnRH is mediated by the two nuclear factor-kappaB sites and the CCAAT/enhancer-binding protein site within its promoter. Indeed, GnRH stimulates p65/RelA phosphorylation (22-fold) in LbetaT2 cells and the two nuclear factor-kappaB sites apparently act as a composite response element. Although GnRH stimulates cAMP formation in LbetaT2 cells, we found no role for cAMP acting via the cAMP response element site in the COX-2 promoter. PGF(2alpha), PGI(2), or PGE(2) had no effect on GnRH-stimulated ERK, c-Jun N-terminal kinase, and p38MAPK activation or on GnRH- and high K(+)-stimulated intracellular Ca(2+) elevation in LbetaT2 and gonadotropes in primary culture. Although, PGF(2alpha), PGI(2), and PGE(2) reduced GnRH-stimulated cAMP formation, we could not correlate it to the inhibition of GnRH receptor expression, which is exerted only by PGF(2alpha) and PGI(2.) Hence, the inhibition by PGF(2alpha) and PGI(2) of the autoregulation of GnRH receptor expression is most likely mediated via inhibition of GnRH-stimulated phosphoinositide turnover and not by inhibition of Ca(2+) elevation and MAPK activation.

651 VERAPAMIL AND PENTOXIFYLLINE INHIBIT TGF-BETA-STIMULATED TIMP-3 PRODUCTION IN PEYRONIE'S DISEASE FIBROBLASTS: EVIDENCE OF CAMP- AND CALCIUM-DEPENDENT SIGNALING CROSSTALK

You have accessJournal of UrologyPenis/Testis/Urethra: Benign & Malignant Disease II1 Apr 2010651 VERAPAMIL AND PENTOXIFYLLINE INHIBIT TGF-BETA-STIMULATED TIMP-3 PRODUCTION IN PEYRONIE'S DISEASE FIBROBLASTS: EVIDENCE OF CAMP- AND CALCIUM-DEPENDENT SIGNALING CROSSTALK Marcello Del Carlo, Ada Cole, and Laurence Levine Marcello Del CarloMarcello Del Carlo More articles by this author , Ada ColeAda Cole More articles by this author , and Laurence LevineLaurence Levine More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1025AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The calcium channel blocker verapamil and the phosphodiesterase type 4 (PDE4) inhibitor pentoxifylline have been reported to be useful as a treatment alternative for patients with Peyronie's Disease (PD). The fibrotic effects of TGF-beta overexpression in PD are well documented but the role of cAMP and calcium as secondary intermediates of TGF-beta signaling is poorly understood. The purpose of this study was to determine the effects of these anti-fibrotic agents on TGF-beta mediated tissue inhibitor of matrix metalloproteinase (TIMP) production as a potential mechanism of action. METHODS Fibroblasts were isolated from fibrotic plaque tissue removed from patients undergoing surgical correction for PD. The effects of verapamil (100 uM) and pentoxifylline (500 uM) on TGF-beta (10 ng/ml) mediated TIMP-3 production was analyzed by immunoblotting conditioned media from monolayer cultures 48 hours after stimulation, and reverse transcription-polymerase chain reaction (RT-PCR) was used to quantify TIMP-3 mRNA in cells 8 hours after stimulation. Intracellular [calcium] fluxes were analyzed by confocal microscopy (lambda-ex = 340/380 nm, lambda-em = 510 nm) using Fura-2/AM (2 uM) loaded cells in the presence and absence of either the PKA activator forskolin (10 uM) or the PKA inhibitor H89 (10 uM) to modulate intracellular [cAMP]. Luciferase reporter constructs were transfected in cells to quantify CREB binding activity. RESULTS Pre-treatment with either verapamil or pentoxifylline reduced TGF-beta mediated TIMP-3 mRNA expression and protein production, and also prevented increased cytosolic calcium fluxes observed in cells stimulated with TGF-beta. Similar results were obtained with the calcium channel blocker nifedipine and the PKA activator forskolin. Pretreatment with either the cell-permeable calcium-chelator BAPTA/AM (5 uM) or the PKA inhibitor H89 had no effect. Treatment with either verapamil or pentoxifylline alone significantly induced CREB activity, which was marginally attenuated in the presence of TGF-beta. CONCLUSIONS Decreased TGF-beta mediated TIMP-3 expression represents an attractive mechanism for the anti-fibrotic effects displayed by verapamil and pentoxifylline therapy in patients with PD. The increased activity of cAMP-sensitive calcium channels caused by TGF-beta overexpression may represent an important biochemical mechanism for the localized fibrosis observed in PD. Chicago, IL© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e255 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Marcello Del Carlo More articles by this author Ada Cole More articles by this author Laurence Levine More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Role of cAMP and cGMP in arachidonic acid and PGE2 ‐induced cerebral vasodilation in newborn pigs

Arachidonic acid (AA) metabolites of cyclooxygenase (COX) are important regulators of newborn cerebral circulation. PGE2 stimulates cAMP in cultured vascular smooth muscle cells. PGE2 increases cAMP and cGMP. Therefore, AA and PGE2, generated in cerebral vessels may produce dilation by increasing cGMP and cAMP. In this study we address the hypothesis that cAMP and/or cGMP mediate dilation produced by AA and PGE2. Experiments used anesthetized newborn pigs with closed cranial windows. In piglets with PGE2, indomethacin (5mg/k g iv) was administered to inhibit COX. Pial arteriolar responses to AA (10−7 M, 10−5 M), PGE2 (10−7 M, 10−5 M) or vehicle with guanylyl cyclase inhibitor ODQ (2.5 x10−5 M) or adenylyl cyclase inhibitor SQ22536 (10−6 M), or their vehicles were determined. ODQ (2.5 x10−5 M) blocked AA‐ and PGE2‐induced increases in cerebral vasodilation in newborn pigs. SQ 22536 blocked dilation produced by the adenylyl cyclase activator forskolin (10−7M) and also inhibited AA‐ and PGE2‐induced cerebral vasodilation. Both cGMP and cAMP appear to contribute to the dilator effect of AA and PGE2. Since both ODQ and SQ22536 blocked dilations to AA and PGE2, cAMP and cGMP may act in a sequential manner. Research supported by NIH/NHLBI.

Effect of amurinone on human pulmonary artery in vitro

We studied the effect of amurinone, a phosphodiesterase inhibitor, on human pulmonary arterial smooth muscle in vitro. Amurinone caused dose-dependent relaxation of pulmonary arterial strips precontracted with 60 mM KCl. Preincubation with either meclofenamate (31 microM), a cyclooxygenase inhibitor, or L-NG-nitroarginine (100 microM), an endothelium-derived relaxing factor (EDRF) production inhibitor, failed to inhibit amurinone-induced pulmonary vasodilation. The cyclic AMP (cAMP) levels in the supernatant of the lung vessel homogenates significantly increased after incubation with amurinone. These results indicate that amurinone causes relaxation of human pulmonary artery in vitro, and suggest a role for cAMP in the mechanisms of amurinone-induced pulmonary vasodilation.

Effects of propofol on β-adrenoceptor-mediated signal transduction in cardiac muscle; role of cAMP

BackgroundPropofol may decrease myocardial contractility via actions on the β-adrenoceptor-mediated signal transduction. The aim of this study was to evaluate the effect of propofol via β-adrenoceptor-mediated signal transduction by measuring the tissue levels of cAMP (cyclic adenosine monophosphate).MethodsThe effects of propofol on β-adrenoceptor mediated cascades were measured with cAMP concentrations, which were stimulated by agonists (l-isoproterenol, GTPγS, and forskolin) of each step of β-adrenoceptor-mediated cascades.ResultsWhile the production of cAMP stimulated by isoproterenol, GTPγS, or forskolin are increased (P < 0.05), application of each concentration of propofol (0.1, 1, 10, 100 µM) did not alter the levels of cAMP.ConclusionsConsidering that propofol did not alter the tissue cAMP levels when stimulated by isoproterenol, GTPγS, and forskolin, propofol appears to have no effect on the β-adrenoceptor signaling pathway in guinea pig ventricular myocardium.

Regulation of Phosphodiesterase 3 in the Pulmonary Arteries During the Perinatal Period in Sheep

The role of cAMP in the pulmonary vasculature during the transition from intrauterine to extrauterine life is poorly understood. We hypothesized that cAMP levels are regulated by alterations in phosphodiesterase 3 (PDE3), which hydrolyzes cAMP. PDE3 protein expression and hydrolytic activity were increased in the resistance pulmonary arteries (PA) from spontaneously breathing 1-d-old (1dSB) lambs relative to equivalent-gestation fetuses. This was accompanied by a decrease in steady-state cAMP. Ventilation with 21% O2 and 100% O2 for 24 h disrupted the normal transition, whereas ventilation with 100% O2 + inhaled NO (iNO) for 24 h restored both PDE3 activity and cAMP to 1dSB levels. Consistent with these findings, relaxation to milrinone, a PDE3 inhibitor, was greater in the PA isolated from 1dSB and 100% O2 + iNO lambs, relative to fetal, 21% O2, and 100% O2 lambs. In conclusion, PDE3 expression and activity in the PA dramatically increase after birth, with a concomitant decrease in steady-state cAMP. Ventilation with either 21% O2 or 100% O2 blunts this PDE3 increase, whereas iNO restores PDE3 activity to levels equivalent to 1dSB lambs. The vasodilatory effects of milrinone were most pronounced in vessels from lambs with the highest PDE3 activity, i.e., 1dSB and 100% O2 + iNO lambs. Thus, milrinone may be most beneficial when used in conjunction with iNO.

The role of cAMP and prostaglandins in gastric acid secretion after pentagastrin administration.

The role of cAMP and prostaglandins as specific intracellular effectors of gastrin action at the level of the parietal cells has not been sufficiently clarified. For this reason we studied the responses of the parietal cells to stimulation with pentagastrin (6 micrograms/kg i.m.) during theophylline infusion (which causes an increase in the intracellular cAMP) and during acetylsalicylic acid infusion (which inhibits the prostaglandin synthesis) in 28 healthy volunteers. Both theophylline and acetylsalicylic acid provoked a significant increase of gastric acid secretion after pentagastrin. Our results suggest that: 1. an increase in intracellular cAMP may be the basis of the stimulatory effect of gastrin on gastric acid secretion 2. a decrease in the synthesis of prostaglandins may lead to a greater gastric acid response after pentagastrin.

Ethanol-Modulated Camouflage Response Screen in Zebrafish Uncovers a Novel Role for cAMP and Extracellular Signal-Regulated Kinase Signaling in Behavioral Sensitivity to Ethanol

Ethanol, a widely abused substance, elicits evolutionarily conserved behavioral responses in a concentration-dependent manner in vivo. The molecular mechanisms underlying such behavioral sensitivity to ethanol are poorly understood. While locomotor-based behavioral genetic screening is successful in identifying genes in invertebrate models, such complex behavior-based screening has proven difficult for recovering genes in vertebrates. Here we report a novel and tractable ethanol response in zebrafish. Using this ethanol-modulated camouflage response as a screening assay, we have identified a zebrafish mutant named fantasma (fan), which displays reduced behavioral sensitivity to ethanol. Positional cloning reveals that fan encodes type 5 adenylyl cyclase (AC5). fan/ac5 is required to maintain the phosphorylation of extracellular signal-regulated kinase (ERK) in the forebrain structures, including the telencephalon and hypothalamus. Partial inhibition of phosphorylation of ERK in wild-type zebrafish mimics the reduction in sensitivity to stimulatory effects of ethanol observed in the fan mutant, whereas, strikingly, strong inhibition of phosphorylation of ERK renders a stimulatory dose of ethanol sedating. Since previous studies in Drosophila and mice show a role of cAMP signaling in suppressing behavioral sensitivity to ethanol, our findings reveal a novel, isoform-specific role of AC signaling in promoting ethanol sensitivity, and suggest that the phosphorylation level of the downstream effector ERK is a critical "gatekeeper" of behavioral sensitivity to ethanol.

Estradiol Increases Guanosine 5′-Triphosphate Cyclohydrolase Expression Via the Nitric Oxide-Mediated Activation of Cyclic Adenosine 5′-Monophosphate Response Element Binding Protein

A number of studies have demonstrated that estradiol can stimulate endothelial nitric oxide synthase expression and activity, resulting in enhanced nitric oxide (NO) generation. However, its effect on the NO synthase cofactor, tetrahydrobiopterin are less clear. Cellular tetrahydrobiopterin levels are regulated, at least in part, by GTP cyclohydrolase 1 (GCH1). Thus, the purpose of this study was to determine the effect of estradiol on GCH1 expression and the regulatory mechanisms in pulmonary arterial endothelial cells. Our data indicate that 17beta-estradiol (E2) increases GCH1 transcription in a dose- and time-dependent manner, whereas estrogen receptor antagonism or NO synthase inhibition attenuated E2-stimulated GCH1 expression. Analysis of the GCH1 promoter fragment responsive to E2 revealed the presence of a cAMP response element, and we found that E2 triggers a rapid but transient elevation of phospho-cAMP response element-binding protein (CREB; <1 h) followed by a second sustained rise after 6 h. EMSA analysis revealed an increase in the binding of CREB during E2 treatment and mutation of the cAMP response element in the GCH1 promoter attenuated the E2-mediated increase in transcription. Furthermore, inhibition of the cAMP-dependent kinase, protein kinase A (PKA) completely abolished the E2-stimulated GCH1 promoter activity, whereas the stimulation of cAMP levels with forskolin increased GCH1 promoter activity, indicating the key role of cAMP in regulating GCH1 promoter activity. In conclusion, our results demonstrate that estradiol can modulate GCH1 expression via NO-mediated activation of CREB in pulmonary arterial endothelial cells. These findings provide new insight into the vascular protective effect of estradiol.

Synergistic IL-10 induction by LPS and the ceramide-1-phosphate analog PCERA-1 is mediated by the cAMP and p38 MAP kinase pathways

Expression of the anti-inflammatory cytokine IL-10 can be induced either by TLR agonists such as lipopolysaccharide (LPS), or by various endogenous stimuli, in particular those acting via a cAMP-dependent signaling pathway. We have previously reported that the synthetic phospho-ceramide analogue-1 (PCERA-1) increases cAMP level and subsequently down-regulates production of TNFα and up-regulates production of IL-10 in LPS-stimulated macrophages. The objective of this study was to determine the mechanism of activity of PCERA-1 and the role of cAMP in LPS-induced IL-10 production. We show here that PCERA-1 induces IL-10 production in synergism with various TLR agonists in mouse RAW264.7 macrophages. Cooperativity is evident both at the mRNA and protein levels. IL-10 production by LPS and PCERA-1 is mediated by the cAMP pathway and by the p38 MAP kinase. Phosphorylation of p38 is cooperatively accomplished by LPS and PCERA-1 or other cAMP inducers. Furthermore, the activity of PCERA-1 can be partially mimicked by a cell-permeable analog of cAMP, and blocked by the protein kinase A (PKA) inhibitor H89. Finally, in the absence of PCERA-1, the residual IL-10 induction by LPS depends on the basal cAMP level as it can be largely elevated by the phosphodiesterase (PDE)-4 inhibitor rolipram. Our results thus indicate that IL-10 induction by LPS critically depends on basal cAMP level, and that a co-stimulus by a TLR agonist and a cAMP-elevating agent results in synergistic PKA-dependent and p38-dependent IL-10 production.

Role of cAMP and cGMP in arachidonic acid and PGE2 ‐induced cerebral vasodilation in piglets

Arachidonic acid (AA) products are important contributors to regulation of newborn cerebral circulation. PGE2 stimulates cAMP production in cultured piglet cerebrovascular smooth muscle cells. PGE2 increases cAMP and cGMP in CSF. Therefore, it is possible that AA and its metabolite, PGE2, generated in cerebral vessels produce vasodilation by increasing cGMP and cAMP production. The aim of this study was to determine if cAMP and/or cGMP mediate the vasodilatory effects of AA and PGE2. Experiments were performed in anesthetized piglets with closed cranial windows. In experiments with PGE2, piglets were administered indomethacin (5mg/kg, iv) to inhibit endogenous prostanoid production. Pial arteriolar responses to topical application of AA (10−7 M, 10−5 M), PGE2 (10−7 M, 10−5 M) or vehicle were determined without and with the guanylyl cyclase inhibitor ODQ (10−5 M) or the adenylyl cyclase inhibitor SQ22536 (10−6 M). ODQ (2.5x10−5 M) blocked AA‐ and PGE2‐induced cerebral vasodilation. SQ22536, that blocked the dilation to the adenylyl cyclase activator, Forskolin (10−7 M), also inhibited AA‐ and PGE2‐induced cerebral vasodilation. These data suggest that both cGMP and cAMP mediate vasodilator effects of AA and PGE2. Since both ODQ and SQ22536 blocked the cerebral vasodilator effect of AA and PGE2, cAMP and cGMP may act in a sequential manner. Studies to address this subject are in progress.