cAMP Regulation Research Articles

Phosphodiesterase Diversity and Signal Processing Within cAMP Signaling Networks.

A large number of neuromodulators activate G-protein coupled receptors (GPCRs) and mediate their cellular actions via the regulation of intracellular cAMP, the small highly diffusible second messenger. In fact, in the same neuron several different GPCRs can regulate cAMP with seemingly identical timecourses that give rise to distinct signaling outcomes, suggesting that cAMP does not have equivalent access to all its downstream effectors and may exist within defined intracellular pools or domains. cAMP compartmentalization is the process that allows the neuron to differentially interpret these various intracellular cAMP signals into cellular response. The molecular mechanisms that give rise to cAMP compartmentalization are not fully understood, but it is thought that phosphodiesterases (PDEs), the enzymes that degrade cAMP, significantly contribute to this process. PDEs, as the sole mechanism of signal termination for cAMP, hold great promise as therapeutic targets for pathologies that are due to the dysregulation of intracellular cAMP signaling. Due to their diverse catalytic activity, regulation and localization each PDE subtype expressed in a given neuron may have a distinct role on downstream signaling.

G-protein-coupled receptor signaling through Gpr176, Gz, and RGS16 tunes time in the center of the circadian clock [Review

G-protein-coupled receptors (GPCRs) constitute an immensely important class of drug targets with diverse clinical applications. There are still more than 120 orphan GPCRs whose cognate ligands and physiological functions are not known. A set of circadian pacemaker neurons that governs daily rhythms in behavior and physiology resides in the suprachiasmatic nucleus (SCN) in the brain. Malfunction of the circadian clock has been linked to a multitude of diseases, such as sleeping disorders, obesity, diabetes, cardiovascular diseases, and cancer, which makes the clock an attractive target for drug development. Here, we review a recently identified role of Gpr176 in the SCN. Gpr176 is an SCN-enriched orphan GPCR that sets the pace of the circadian clock in the SCN. Even without known ligand, this orphan receptor has an agonist-independent basal activity to reduce cAMP signaling. A unique cAMP-repressing G-protein subclass Gz is required for the activity of Gpr176. We also provide an overview on the circadian regulation of G-protein signaling, with an emphasis on a role for the regulator of G-protein signaling 16 (RGS16). RGS16 is indispensable for the circadian regulation of cAMP in the SCN. Developing drugs that target the SCN remains an unfulfilled opportunity for the circadian pharmacology. This review argues for the potential impact of focusing on GPCRs in the SCN for the purpose of tuning the body clock.

Newly synthesized cAMP is integrated at a membrane protein complex signalosome to ensure receptor response specificity.

Spatiotemporal regulation of cAMP within the cell is required to achieve receptor-specific responses. The mechanism through which the cell selects a specific response to newly synthesized cAMP is not fully understood. In hepatocyte plasma membranes, we identified two functional and independent cAMP-responsive signaling protein macrocomplexes that produce, use, degrade, and regulate their own nondiffusible (sequestered) cAMP pool to achieve their specific responses. Each complex responds to the stimulation of an adenosine G protein-coupled receptor (Ado-GPCR), bound to either A2A or A2B , but not simultaneously to both. Each isoprotein involved in each signaling cascade was identified by measuring changes in cAMP levels after receptor activation, and its participation was confirmed by antibody-mediated inactivation. A2A -Ado-GPCR selective stimulation activates adenylyl cyclase 6 (AC6), which is bound to AKAP79/150, to synthesize cAMP which is used by two other AKAP79/150-tethered proteins: protein kinase A (PKA) and phosphodiesterase 3A (PDE3A). In contrast, A2B -Ado-GPCR stimulation activates D-AKAP2-attached AC5 to generate cAMP, which is channeled to two other D-AKAP2-tethered proteins: guanine-nucleotide exchange factor 2 (Epac2) and PDE3B. In both cases, prior activation of PKA or Epac2 with selective cAMP analogs prevents de novo cAMP synthesis. In addition, we show that cAMP does not diffuse between these protein macrocomplexes or 'signalosomes'. Evidence of coimmunoprecipitation and colocalization of some proteins belonging to each signalosome is presented. Each signalosome constitutes a minimal functional signaling unit with its own machinery to synthesize and regulate a sequestered cAMP pool. Thus, each signalosome is devoted to ensure the transmission of a unique and unequivocal message through the cell.

Model-guided optogenetic study of PKA signaling in budding yeast.

In eukaryotes, protein kinase A (PKA) is a master regulator of cell proliferation and survival. The activity of PKA is subject to elaborate control and exhibits complex time dynamics. To probe the quantitative attributes of PKA dynamics in the yeast Saccharomyces cerevisiae, we developed an optogenetic strategy that uses a photoactivatable adenylate cyclase to achieve real-time regulation of cAMP and the PKA pathway. We capitalize on the precise and rapid control afforded by this optogenetic tool, together with quantitative computational modeling, to study the properties of feedback in the PKA signaling network and dissect the nonintuitive dynamic effects that ensue from perturbing its components. Our analyses reveal that negative feedback channeled through the Ras1/2 GTPase is delayed, pinpointing its time scale and its contribution to the dynamic features of the cAMP/PKA signaling network.

Cilia have high cAMP levels that are inhibited by Sonic Hedgehog-regulated calcium dynamics

Protein kinase A (PKA) phosphorylates Gli proteins, acting as a negative regulator of the Hedgehog pathway. PKA was recently detected within the cilium, and PKA activity specifically in cilia regulates Gli processing. Using a cilia-targeted genetically encoded sensor, we found significant basal PKA activity. Using another targeted sensor, we measured basal ciliary cAMP that is fivefold higher than whole-cell cAMP. The elevated basal ciliary cAMP level is a result of adenylyl cyclase 5 and 6 activity that depends on ciliary phosphatidylinositol (3,4,5)-trisphosphate (PIP3), not stimulatory G protein (Gαs), signaling. Sonic Hedgehog (SHH) reduces ciliary cAMP levels, inhibits ciliary PKA activity, and increases Gli1. Remarkably, SHH regulation of ciliary cAMP and downstream signals is not dependent on inhibitory G protein (Gαi/o) signaling but rather Ca2+ entry through a Gd3+-sensitive channel. Therefore, PIP3 sustains high basal cAMP that maintains PKA activity in cilia and Gli repression. SHH activates Gli by inhibiting cAMP through a G protein-independent mechanism that requires extracellular Ca2+ entry.

Modulation of Compartmentalised Cyclic Nucleotide Signalling via Local Inhibition of Phosphodiesterase Activity.

Cyclic nucleotide phosphodiesterases (PDEs) are the only enzymes that degrade the cyclic nucleotides cAMP and cGMP, and play a key role in modulating the amplitude and duration of the signal delivered by these two key intracellular second messengers. Defects in cyclic nucleotide signalling are known to be involved in several pathologies. As a consequence, PDEs have long been recognized as potential drug targets, and they have been the focus of intense research for the development of therapeutic agents. A number of PDE inhibitors are currently available for the treatment of disease, including obstructive pulmonary disease, erectile dysfunction, and heart failure. However, the performance of these drugs is not always satisfactory, due to a lack of PDE-isoform specificity and their consequent adverse side effects. Recent advances in our understanding of compartmentalised cyclic nucleotide signalling and the role of PDEs in local regulation of cAMP and cGMP signals offers the opportunity for the development of novel strategies for therapeutic intervention that may overcome the current limitation of conventional PDE inhibitors.

Differential Expression and Function of PDE8 and PDE4 in Effector T cells: Implications for PDE8 as a Drug Target in Inflammation.

Abolishing the inhibitory signal of intracellular cAMP is a prerequisite for effector T (Teff) cell function. The regulation of cAMP within leukocytes critically depends on its degradation by cyclic nucleotide phosphodiesterases (PDEs). We have previously shown that PDE8A, a PDE isoform with 40–100-fold greater affinity for cAMP than PDE4, is selectively expressed in Teff vs. regulatory T (Treg) cells and controls CD4+ Teff cell adhesion and chemotaxis. Here, we determined PDE8A expression and function in CD4+ Teff cell populations in vivo. Using magnetic bead separation to purify leukocyte populations from the lung draining hilar lymph node (HLN) in a mouse model of ovalbumin-induced allergic airway disease (AAD), we found by Western immunoblot and quantitative (q)RT-PCR that PDE8A protein and gene expression are enhanced in the CD4+ T cell fraction over the course of the acute inflammatory disease and recede at the late tolerant non-inflammatory stage. To evaluate PDE8A as a potential drug target, we compared the selective and combined effects of the recently characterized highly potent PDE8-selective inhibitor PF-04957325 with the PDE4-selective inhibitor piclamilast (PICL). As previously shown, PF-04957325 suppresses T cell adhesion to endothelial cells. In contrast, we found that PICL alone increased firm T cell adhesion to endothelial cells by ~20% and significantly abrogated the inhibitory effect of PF-04957325 on T cell adhesion by over 50% when cells were co-exposed to PICL and PF-04957325. Despite its robust effect on T cell adhesion, PF-04957325 was over two orders of magnitude less efficient than PICL in suppressing polyclonal Teff cell proliferation, and showed no effect on cytokine gene expression in these cells. More importantly, PDE8 inhibition did not suppress proliferation and cytokine production of myelin-antigen reactive proinflammatory Teff cells in vivo and in vitro. Thus, targeting PDE8 through PF-04957325 selectively regulates Teff cell interactions with endothelial cells without marked immunosuppression of proliferation, while PDE4 inhibition has partially opposing effects. Collectively, our data identify PF-04957325 as a novel function-specific tool for the suppression of Teff cell adhesion and indicate that PDE4 and PDE8 play unique and non-redundant roles in the control of Teff cell functions.

Effects of a novel phosphodiesterase 10A inhibitor in non-human primates: A therapeutic approach for schizophrenia with improved side effect profile

Schizophrenia symptoms are associated with alterations in basal ganglia-cortical networks that include the cyclic nucleotides (cAMP/cGMP) signaling pathways. Phosphodiesterase 10A (PDE10A) inhibitors have been considered as therapeutic agents for schizophrenia because the regulation of cAMP and cGMP in the striatum by PDE10A plays an important role in the signaling mechanisms of the striatal-cortical network, and thereby in cognitive function. In the present study we assessed in non-human primates (NHPs) the effects of a novel PDE10A inhibitor (FRM-6308) that has demonstrated high potency and selectivity for human recombinant PDE10A in vitro. The behavioral effects of FRM-6308 in a dose range were determined in rhesus monkeys using a standardized motor disability scale for primates, motor tasks, and the “drug effects on the nervous system” (DENS) scale. The neuronal metabolic effects of FRM-6308 were determined with [(18)F]-fluorodeoxyglucose PET imaging. Results showed that FRM-6308 did not have any specific effects on the motor system at s.c. doses up to 0.32 mg/kg in NHPs, which induced a significant increase in the FDG-SUV in striatum (F 16.069, p < 0.05) and cortical (F 15.181, p < 0.05) regions. Higher doses induced sedation and occasional involuntary movements with clear development of tolerance after repeated exposures. These findings suggest that FRM-6308 has the adequate pharmacological profile to advance testing in clinical trials and demonstrate antipsychotic efficacy of PDE10A inhibition for the treatment of schizophrenia patients.

Cyclic nucleotide regulation of cardiac sympatho-vagal responsiveness.

Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are now recognized as important intracellular signalling molecules that modulate cardiac sympatho-vagal balance in the progression of heart disease. Recent studies have identified that a significant component of autonomic dysfunction associated with several cardiovascular pathologies resides at the end organ, and is coupled to impairment of cyclic nucleotide targeted pathways linked to abnormal intracellular calcium handling and cardiac neurotransmission. Emerging evidence also suggests that cyclic nucleotide coupled phosphodiesterases (PDEs) play a key role limiting the hydrolysis of cAMP and cGMP in disease, and as a consequence this influences the action of the nucleotide on its downstream biological target. In this review, we illustrate the action of nitric oxide-CAPON signalling and brain natriuretic peptide on cGMP and cAMP regulation of cardiac sympatho-vagal transmission in hypertension and ischaemic heart disease. Moreover, we address how PDE2A is now emerging as a major target that affects the efficacy of soluble/particulate guanylate cyclase coupling to cGMP in cardiac dysautonomia.

Skeletal Muscle Gene Expression In Elite Ultra-endurance Athletes Habitually Consuming Very Low-carbohydrate or High-carbohydrate Diets

A growing number of elite-level ultra-endurance athletes have switched from a high-carbohydrate (HCD) to a very low-carbohydrate/high-fat diet (LCD). LCD athletes exhibit greater than two-fold higher rates of peak fat oxidation compared to their high-carbohydrate counterparts. PURPOSE: In order to determine if the fat-adapted metabolic phenotype was associated with differences in skeletal muscle gene expression, we performed genome wide transcriptomic analysis in athletes who had been habitually consuming a LCD for a minimum of six months. METHODS: 20 elite ultra-endurance athletes (men, age 33.5 ± 6.4 yr, BMI 22.6 ± 3.3 kg/m2, VO2max 64.5 ± 4.9 mL/kg/min) habitually consuming a HCD (n=10; 58% CHO, 15% PRO, 28% FAT) or a LCD (n=10; 11% CHO, 19% PRO, 71% FAT) were matched for age, competition events, and performance. Muscle biopsies from the vastus lateralis were obtained in a fasted state. cDNA library was prepared from total RNA and sequenced for transcriptome expression using the Illumina HiSeq2500. Reads per kilobase of transcripts per million analysis was used to compare gene expression between groups (p ≤ 0.01) and with absolute fold changes (±2). RESULTS: Fat-adapted athletes had an average of 20 months (range 9-36 mo) on a LCD. Of the 25,262 total genes sequenced, 633 showed dietary differences (p ≤ 0.01). Sixty genes had greater than a 2-fold magnitude change including 47 upregulated (p ≤ 0.01) and 13 downregulated (p ≤ 0.01) genes. In the LCD athletes, two of the most significant upregulated genes coded for protein phosphatase 1 regulatory subunit 1A (+2.2 fold), an inhibitor of glycogen metabolism, and 3-hydroxy-3-methylglutaryl-CoA synthase 2 (+19.6 fold), an enzyme in ketogensis. The most significant downregulated gene coded for cAMP-specific 3’,5’-cyclic phosphodiesterase 4C, a key regulator of cAMP. CONCLUSION: Athletes habitually following a ketogenic diet showed distinct gene expression patterns that may give insight into the molecular mechanisms that mediate the fat-adapted phenotype.

Spatiotemporal Dynamics and Regulation of cAMP and PKA in Early Stage Hippocampal Neurons

Cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) are critical axon determinants that regulate neuronal polarity and axon formation, but the spatiotemporal dynamics of cAMP and PKA in developing neuronal subcompartments are poorly understood. Here, we use genetically encoded fluorescent biosensors to understand the compartmentalized dynamics of cAMP and PKA signaling in developing hippocampal neurons in vitro. In five‐day‐old hippocampal neurons, we detected an axon‐directed cAMP gradient dependent on phosphodiesterase activity and an axon‐directed PKA gradient dependent on the association of PKA with A‐kinase anchoring proteins (AKAPs). Furthermore, disrupting PKA anchoring to AKAPs in early hippocampal neurons leads to enhanced axon elongation. This effect is correlated with an enhancement of the stimulated axon‐directed cAMP and PKA gradients. We propose that a negative feedback loop in the cAMP/PKA/AKAP signaling microdomain plays an essential role in controlling the developmental program of hippocampal neurons.Support or Funding InformationThis work was supported by NIH RO1 DK073368 (to J.Z.), DGIST Convergence Science Center grant 11‐BD‐04 (to G.V.R), NSF GRF 1232825 (to K.G.).

The Odorant Receptor-Dependent Role of Olfactory Marker Protein in Olfactory Receptor Neurons.

Olfactory receptor neurons (ORNs) in the nasal cavity detect and transduce odorants into action potentials to be conveyed to the olfactory bulb. Odorants are delivered to ORNs via the inhaled air at breathing frequencies that can vary from 2 to 10 Hz in the mouse. Thus olfactory transduction should occur at sufficient speed such that it can accommodate repetitive and frequent stimulation. Activation of odorant receptors (ORs) leads to adenylyl cyclase III activation, cAMP increase, and opening of cyclic nucleotide-gated channels. This makes the kinetic regulation of cAMP one of the important determinants for the response time course. We addressed the dynamic regulation of cAMP during the odorant response and examined how basal levels of cAMP are controlled. The latter is particularly relevant as basal cAMP depends on the basal activity of the expressed OR and thus varies across ORNs. We found that olfactory marker protein (OMP), a protein expressed in mature ORNs, controls both basal and odorant-induced cAMP levels in an OR-dependent manner. Lack of OMP increases basal cAMP, thus abolishing differences in basal cAMP levels between ORNs expressing different ORs. Moreover, OMP speeds up signal transduction for ORNs to better synchronize their output with high-frequency stimulation and to perceive brief stimuli. Last, OMP also steepens the dose-response relation to improve concentration coding although at the cost of losing responses to weak stimuli. We conclude that OMP plays a key regulatory role in ORN physiology by controlling multiple facets of the odorant response.

Transcription factor CREB3L1 mediates cAMP and glucocorticoid regulation of arginine vasopressin gene transcription in the rat hypothalamus.

BackgroundArginine vasopressin (AVP), a neuropeptide hormone that functions in the regulation of water homeostasis by controlling water re-absorption at kidneys, is synthesised in supraoptic nucleus and paraventricular nucleus of the hypothalamus. An increase in plasma osmolality stimulates secretion of AVP to blood circulation and induces AVP synthesis in these nuclei. Although studies on mechanism of AVP transcriptional regulation in hypothalamus proposed that cAMP and glucocorticoids positively and negatively regulate Avp expression, respectively, the molecular mechanisms have remained elusive. Recently, we identified CREB3L1 (cAMP-responsive element binding protein 3 like 1) as a putative transcription factor of Avp transcription in the rat hypothalamus. However the mechanism of how CREB3L1 is regulated in response of hyperosmotic stress in the neurons of hypothalamus has never been reported. This study aims to investigate effect of previously reported regulators (cAMP and glucocorticoid) of Avp transcription on transcription factor CREB3L1 in order to establish a molecular explanation for cAMP and glucocorticoids effect on AVP expression.ResultsThe effect of cAMP and glucocorticoid treatment on Creb3l1 was investigated in both AtT20 cells and hypothalamic organotypic cultures. The expression of Creb3l1 was increased in both mRNA and protein level by treatment with forskolin, which raises intracellular cAMP levels. Activation of cAMP by forskolin also increased Avp promoter activity in AtT20 cells and this effect was blunted by shRNA mediated silencing of Creb3l1. The forskolin induced increase in Creb3l1 expression was diminished by combined treatment with dexamethasone, and, in vivo, intraperitoneal dexamethasone injection blunted the increase in Creb3l1 and Avp expression induced by hyperosmotic stress.ConclusionHere we shows that cAMP and glucocorticoid positively and negatively regulate Creb3l1 expression in the rat hypothalamus, respectively, and regulation of cAMP on AVP expression is mediated through CREB3L1. This data provides the connection between CREB3L1, a newly identified transcription factor of AVP expression, with the previously proposed mechanism of Avp transcription which extends our understanding in transcription regulation of Avp in the hypothalamus.

Phosphoinositide 3-Kinase γ Restrains Neurotoxic Effects of Microglia After Focal Brain Ischemia.

Phosphoinositide 3-kinase γ (PI3Kγ) is linked to neuroinflammation and phagocytosis. This study was conducted to elucidate conjectural differences of lipid kinase-dependent and kinase-independent functions of PI3Kγ in the evolvement of brain damage induced by focal cerebral ischemia/reperfusion. Therefore, PI3Kγ wild-type, knockout, and kinase-dead mice were subjected to middle cerebral artery occlusion followed by reperfusion. Tissue damage and cellular composition were assessed by immunohistochemical stainings. In addition, microglial cells derived from respective mouse genotypes were used for analysis of PI3Kγ effects on phagocytic activity, matrix metalloproteinase-9 release, and cAMP content under conditions of oxygen/glucose deprivation and recovery. Brain infarction was more pronounced in PI3Kγ-knockout mice compared to wild-type and kinase-dead mice 48h after reperfusion. Immunohistochemical analyses revealed a reduced amount of galectin-3/MAC-2-positive microglial cells indicating that activated phagocytosis was reduced in ischemic brains of knockout mice. Cell culture studies disclosed enhanced metalloproteinase-9 secretion in supernatants derived from microglia of PI3Kγ-deficient mice after 2-h oxygen/glucose deprivation and 48-h recovery. Furthermore, PI3Kγ-deficient microglial cells showed a failed phagocytic activation throughout the observed recovery period. Lastly, PI3Kγ-deficient microglia exhibited strongly increased cAMP levels in comparison with wild-type microglia or cells expressing kinase-dead PI3Kγ after oxygen/glucose deprivation and recovery. Our data suggest PI3Kγ kinase activity-independent control of cAMP phosphodiesterase as a crucial mediator of microglial cAMP regulation, MMP-9 expression, and phagocytic activity following focal brain ischemia/recirculation. The suppressive effect of PI3Kγ on cAMP levels appears critical for the restriction of ischemia-induced immune cell functions and in turn tissue damage.

The ever unfolding story of cAMP signaling in trypanosomatids: vive la difference!

Kinetoplastids are unicellular, eukaryotic, flagellated protozoans containing the eponymous kinetoplast. Within this order, the family of trypanosomatids are responsible for some of the most serious human diseases, including Chagas disease (Trypanosoma cruzi), sleeping sickness (Trypanosoma brucei spp.), and leishmaniasis (Leishmania spp). Although cAMP is produced during the life cycle stages of these parasites, its signaling pathways are very different from those of mammals. The absence of G-protein-coupled receptors, the presence of structurally different adenylyl cyclases, the paucity of known cAMP effector proteins and the stringent need for regulation of cAMP in the small kinetoplastid cells all suggest a significantly different biochemical pathway and likely cell biology. However, each of the main kinetoplastid parasites express four class 1-type cyclic nucleotide-specific phosphodiesterases (PDEA-D), which have highly similar catalytic domains to that of human PDEs. To date, only TbrPDEB, expressed as two slightly different isoforms TbrPDEB1 and B2, has been found to be essential when ablated. Although the genomes contain reasonably well conserved genes for catalytic and regulatory domains of protein kinase A, these have been shown to have varied structural and functional roles in the different species. Recent discovery of a role of cAMP/AMP metabolism in a quorum-sensing signaling pathway in T. brucei, and the identification of downstream cAMP Response Proteins (CARPs) whose expression levels correlate with sensitivity to PDE inhibitors, suggests a complex signaling cascade. The interplay between the roles of these novel CARPs and the quorum-sensing signaling pathway on cell division and differentiation makes for intriguing cell biology and a new paradigm in cAMP signal transduction, as well as potential targets for trypanosomatid-specific cAMP pathway-based therapeutics.

Phosphodiesterase Isoform Regulation of Cell Proliferation and Fluid Secretion in Autosomal Dominant Polycystic Kidney Disease.

cAMP stimulates cell proliferation and Cl(-)-dependent fluid secretion, promoting the progressive enlargement of renal cysts in autosomal dominant polycystic kidney disease (ADPKD). Intracellular cAMP levels are determined by the balance of cAMP synthesis by adenylyl cyclases and degradation by phosphodiesterases (PDEs). Therefore, PDE isoform expression and activity strongly influence global and compartmentalized cAMP levels. We report here that PDE3 and PDE4 expression levels are lower in human ADPKD tissue and cells compared with those of normal human kidneys (NHKs), whereas PDE1 levels are not significantly different. Inhibition of PDE4 caused a greater increase in basal and vasopressin (AVP)-stimulated cAMP levels and Cl(-) secretion by ADPKD cells than inhibition of PDE1, and inhibition of PDE4 induced cyst-like dilations in cultured mouse Pkd1(-/-) embryonic kidneys. In contrast, inhibition of PDE1 caused greater stimulation of extracellular signal-regulated kinase (ERK) and proliferation of ADPKD cells than inhibition of PDE4, and inhibition of PDE1 enhanced AVP-induced ERK activation. Notably, inhibition of PDE1, the only family of Ca(2+)-regulated PDEs, also induced a mitogenic response to AVP in NHK cells, similar to the effect of restricting intracellular Ca(2+). PDE1 coimmunoprecipitated with B-Raf and A-kinase anchoring protein 79, and AVP increased this interaction in ADPKD but not NHK cells. These data suggest that whereas PDE4 is the major PDE isoform involved in the regulation of global intracellular cAMP and Cl(-) secretion, PDE1 specifically affects the cAMP signal to the B-Raf/MEK/ERK pathway and regulates AVP-induced proliferation of ADPKD cells.

Rethinking the roles of CRP, cAMP, and sugar-mediated global regulation in the Vibrionaceae.

Many proteobacteria modulate a suite of catabolic genes using the second messenger cyclic 3', 5'-AMP (cAMP) and the cAMP receptor protein (CRP). Together, the cAMP-CRP complex regulates target promoters, usually by activating transcription. In the canonical model, the phosphotransferase system (PTS), and in particular the EIIA(Glc) component for glucose uptake, provides a mechanistic link that modulates cAMP levels depending on glucose availability, resulting in more cAMP and activation of alternative catabolic pathways when glucose is unavailable. Within the Vibrionaceae, cAMP-CRP appears to play the classical role in modulating metabolic pathways; however, it also controls functions involved in natural competence, bioluminescence, pheromone signaling, and colonization of animal hosts. For this group of marine bacteria, chitin is an ecologically relevant resource, and chitin's monomeric sugar N-acetylglucosamine (NAG) supports robust growth while also triggering regulatory responses. Recent studies with Vibrio fischeri indicate that NAG and glucose uptake share EIIA(Glc), yet the responses of cAMP-CRP to these two carbon sources are starkly different. Moreover, control of cAMP levels appears to be more dominantly controlled by export and degradation. Perhaps more surprisingly, although CRP may require cAMP, its activity can be controlled in response to glucose by a mechanism independent of cAMP levels. Future studies in this area promise to shed new light on the role of cAMP and CRP.

Targeting brain tumor cAMP: the case for sex-specific therapeutics.

A relationship between cyclic adenosine 3′, 5′-monophosphate (cAMP) levels and brain tumor biology has been evident for nearly as long as cAMP and its synthetase, adenylate cyclase (ADCY) have been known. The importance of the pathway in brain tumorigenesis has been demonstrated in vitro and in multiple animal models. Recently, we provided human validation for a cooperating oncogenic role for cAMP in brain tumorigenesis when we found that SNPs in ADCY8 were correlated with glioma (brain tumor) risk in individuals with Neurofibromatosis type 1 (NF1). Together, these studies provide a strong rationale for targeting cAMP in brain tumor therapy. However, the cAMP pathway is well-known to be sexually dimorphic, and SNPs in ADCY8 affected glioma risk in a sex-specific fashion, elevating the risk for females while protecting males. The cAMP pathway can be targeted at multiple levels in the regulation of its synthesis and degradation. Sex differences in response to drugs that target cAMP regulators indicate that successful targeting of the cAMP pathway for brain tumor patients is likely to require matching specific mechanisms of drug action with patient sex.

Adrenomedullin in Vascular Endothelial Injury and Combination Therapy: Time for a New Paradigm.

Adrenomedullin is a highly conserved vasoactive peptide participating in a variety of physiological and pathophysiological processes including pregnancy, embryonic development, tumor progression and regulation of vascular tone. In particular, adrenomedullin expression is widely distributed throughout the cardiovascular system including heart, lungs, blood vessels and kidneys. The observation that adrenomedullin co-localizes with its receptors suggests that the peptide may act as an autocrine and/or paracrine factor in the regulation of cardiovascular function. In this review, we described the pathophysiological changes in plasma and local adrenomedullin associated with vascular endothelial function and the signaling mechanisms involved. Recent evidence has depicted a unique role of adrenomedullin in the regulation of vascular endothelial function in part through regulation of cAMP, cGMP and intracellular Ca(2+) mobilization. This review summarized vascular endothelial actions of adrenomedullin under both physiological and pathophysiological conditions in an effort to provide guidance for the clinical application of adrenomedullin in vascular diseases.

Dopamine receptor D4 internalization requires a beta-arrestin and a visual arrestin

PurposeThe G-protein coupled receptor (GPCR) Dopamine Receptor D4 (DRD4) plays an essential role in cAMP regulation and gap junctional coupling in the photoreceptors, where DRD4 expression is under circadian control. Previous in vitro transfection studies of human DRD4 desensitization have reported that DRD4 is not internalized upon dopamine stimulation when beta-arrestin is co-transfected with DRD4. We hypothesized that the visual arrestins, ARR1 and ARR4, play a modulatory role in DRD4 desensitization in the photoreceptors. MethodsTo test this hypothesis, immunohistochemistry analysis of mouse retinas was used to determine the cellular localization of beta-arrestins and DRD4 in photoreceptors. In vitro studies were performed in HEK293T cells transiently transfected with human DRD4 and arrestins. First, co-immunoprecipitation experiments were executed to test protein–protein interactions and to investigate the effect of dopamine stimulation. Second, immunohistochemistry analysis was implemented to study DRD4 internalization and translocation of ARR4. ResultsImmunohistochemistry studies of mouse retinas confirmed the expression of beta-arrestin 2, ARR1 and ARR4, as well as DRD4 in mouse cone photoreceptor inner segments. Co-immunoprecipitation experiments revealed a dopamine-dependent protein–protein interaction between human DRD4 and ARR4. In vitro internalization experiments showed that no detectable internalization of DRD4 was observed with any single arrestin co-transfected. However, a dopamine-dependent internalization of DRD4 was observed with three out of six sets of two arrestins co-transfected with DRD4. Each of these pairs of arrestins contained one visual arrestin and one beta-arrestin, and no internalization was observed with either two visual arrestins or two beta-arrestins. Additional time-course experiments revealed that in vitro, ARR4 translocates to co-localize with DRD4 at the plasma membrane in response to 30min of dopamine stimulation. ConclusionsThe results have functional implications and we hypothesize that the desensitization and internalization of DRD4 in photoreceptors are synergistically mediated by both visual and beta-arrestins. These results are additionally unique because they demonstrate for the first time that at least one G-protein coupled receptor, DRD4, requires two arrestins for desensitization and internalization, and opens up the possibility that other G-protein coupled receptors may require more than one arrestin for desensitization and/or internalization.