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1. Rat cultured aortic vascular smooth muscle cells (VSMC) express both cyclic GMP-inhibited cyclic AMP phosphodiesterase (PDE3) and Ro 20-1724-inhibited cyclic AMP phosphodiesterase (PDE4) activities. By utilizing either cilostamide, a PDE3-selective inhibitor, or Ro 20-1724, a PDE4-selective inhibitor, PDE3 and PDE4 activities were shown to account for 15% and 55% of total VSMC cyclic AMP phosphodiesterase (PDE) activity. 2. Treatment of VSMC with either forskolin or 8-bromo-cyclic AMP caused significant concentration- and time-dependent increases in total cellular cyclic AMP PDE activity. Using cilostamide or Ro 20-1724, we demonstrated that both PDE3 and PDE4 activities were increased following forskolin or 8-bromo-cyclic AMP treatment, with a relatively larger effect observed on PDE3 activity. The increase in cyclic AMP PDE activity induced by forskolin or 8-bromo-cyclic AMP was inhibited by actinomycin D or cycloheximide, demonstrating that new mRNA synthesis and protein synthesis were required. An analogue of forskolin which does not activate adenylyl cyclase (1,9-dideoxyforskolin) or an analogue of cyclic GMP (8-bromo-cyclic GMP) did not affect total cyclic AMP PDE activity. 3. Incubation of VSMC with 8-bromo-cyclic AMP for 16 h caused a marked rightward shift in the concentration-response curves for both isoprenaline- and forskolin-mediated activation of adenylyl cyclase. A role for up-regulated cyclic AMP PDE activity in this reduced potency is supported by our observation that cyclic AMP PDE inhibitors (IBMX, cilostamide or Ro 20-1724) partially normalized the effects of isoprenaline or forskolin in treated cells to those in untreated cells. 4. We conclude that VSMC cyclic AMP PDE activity is increased following long-term elevation of cyclic AMP and that increases in PDE3 and PDE4 activities account for more than 70% of this effect. Furthermore, we conclude that increases in cyclic AMP PDE activity contribute to the reduced potency of isoprenaline or forskolin in treated VSMC. These results have implications for long-term use of cyclic AMP PDE inhibitors as therapeutic agents.
Escherichia coli cyclic-AMP receptor protein (CRP) represents one of the paradigms of bacterial gene regulation. Yet despite decades of intensive study, new information continues to emerge that prompts reassessment of this classic regulatory system. Moreover, in recent years CRPs from several other bacterial species have been characterized, allowing the general applicability of the CRP paradigm to be tested. Here the properties of the E. coli, Mycobacterium tuberculosis and Pseudomonas putida CRPs are considered in the context of the ecological niches occupied by these bacteria. It appears that the cyclic-AMP-CRP regulatory system has been adapted to respond to distinct external and internal inputs across a broad sensitivity range that is, at least in part, determined by bacterial lifestyles.
Second messenger signaling is required for cellular processes. We previously reported that extracellular vesicles (EVs) from stimulated cultured endothelial cells contain the biochemical second messenger, cAMP. In the current study, we sought to determine whether cAMP-enriched EVs induce second messenger signaling pathways in naïve recipient cells. Our results indicate that cAMP-enriched EVs increase cAMP content sufficient to stimulate PKA activity. The implications of our work are that EVs represent a novel intercellular mechanism for second messenger, specifically cAMP, signaling.
Cell signaling relies extensively on dynamic pools of redox-inactive metal ions such as sodium, potassium, calcium and zinc, but their redox-active transition metal counterparts such as copper and iron have been studied primarily as static enzyme cofactors. Here we report that copper is an endogenous regulator of lipolysis, the breakdown of fat, which is an essential process in maintaining body weight and energy stores. Using a mouse model of genetic copper misregulation, in combination with pharmacological alterations in copper status and imaging studies in a 3T3-L1 white adipocyte model, we found that copper regulates lipolysis at the level of the second messenger, cyclic AMP (cAMP), by altering the activity of the cAMP-degrading phosphodiesterase PDE3B. Biochemical studies of the copper-PDE3B interaction establish copper-dependent inhibition of enzyme activity and identify a key conserved cysteine residue in a PDE3-specific loop that is essential for the observed copper-dependent lipolytic phenotype.
1. The involvement of cyclic AMP-dependent protein kinase (PKA) and cyclic GMP-dependent protein kinase (PKC) in the effects of cyclic AMP-elevating agents on vascular smooth muscle relaxation, cyclic nucleotide dependent-protein kinase activities and ATP-induced calcium signalling ([Ca2+]i was studied in rat aorta. Cyclic AMP-elevating agents used were a beta-adrenoceptor agonist (isoprenaline), a phosphodiesterase 3 (PDE3) inhibitor (SK&F 94120) and a PDE4 inhibitor (rolipram). 2. In rat intact aorta, the relaxant effect induced by isoprenaline (0.01-0.03 microM) was decreased by a specific inhibitor of PKA, H-89, whereas a specific inhibitor of PKG, Rp-8-Br-cyclic GMPs, was without effect. NO significant difference in PKA and PKG activity ratios was detected in aortic rings when isoprenaline 10 microM was used. At the same concentration, isoprenaline did not modify ATP-induced changes in [Ca2+]i in smooth muscle cells. Neither H-89 nor Rp-8-Br-cyclic GMPs modified this response. These findings suggest that PKA is only involved in the relaxant effect induced by low concentrations of isoprenaline (0.01-0.3 microM), whereas for higher concentrations, other mechanisms independent of PKA and PKG were involved. 3. The relaxant effects induced by SK&F 94120 and rolipram were inhibited by Rp-8-Br-cyclic GMPS with no significant effect of H-89. Neither SK&F 94120, nor rolipram at 30 microM significantly modified the activity ratios of PKA and PKG. Rolipram inhibited the ATP-induced transient increase in [Ca2+]i. This decrease was abolished by Rp-8-Br-cyclic GMPS whereas H-89 had no significant effect. These results suggests that PKG is involved in the vascular effects induced by the inhibitors of PDE3 and PDE4. Moreover, since it was previously shown that PDE3 and PDE4 inhibitors only increased cyclic AMP levels with no change in cyclic GMP level, these data also suggest a cross-activation of PKG by cyclic AMP in rat aorta. 4. The combinations of 5 microM SK&F 94120 with rolipram markedly potentiated the relaxant effect of rolipram. This relaxation was decreased by H-89 and not significantly modified by Rp-8-Br-cyclic GMPS. Moreover, the association of the two PDE inhibitors significantly increased the activity ratio of PKA without changing the PKG ratio. The present findings show that PKA rather than PKG is involved in this type of vasorelaxation. The differences in the participation of PKA vs PKG observed when inhibitors of PDE3 and PDE4 were used alone or together could be due to differences in the degree of accumulation of cyclic AMP, resulting in the activation of PKA or PKG which are differently localized in the cell. 5. These findings support for both PKA and PKG in cyclic AMP-mediated relaxation in raT aorta. Their involvement depends on the cellular pathway used to increase the cyclic AMP level.
Platelet activation contributes to normal haemostasis but also to pathologic conditions like stroke and cardiac infarction. Signalling by cGMP and cAMP inhibit platelet activation and are therefore attractive targets for thrombosis prevention. However, extensive cross-talk between the cGMP and cAMP signalling pathways in multiple tissues complicates the selective targeting of their activities. We have used mathematical modelling based on experimental data from the literature to quantify the steady state behaviour of nitric oxide (NO)/cGMP/cAMP signalling in platelets. The analysis provides an assessment of NO-induced cGMP synthesis and PKG activation as well as cGMP-mediated cAMP and PKA activation though modulation of phosphodiesterase (PDE2 and 3) activities. Both one- and two-compartment models of platelet cyclic nucleotide signalling are presented. The models provide new insight for understanding how NO signalling to cGMP and indirectly cAMP, can inhibit platelet shape-change, the initial step of platelet activation. Only the two-compartment models could account for the experimental observation that NO-mediated PKA activation can occur when the bulk platelet cAMP level is unchanged. The models revealed also a potential for hierarchical interplay between the different platelet phosphodiesterases. Specifically, the models predict, unexpectedly, a strong effect of pharmacological inhibitors of cGMP-specific PDE5 on the cGMP/cAMP cross-talk. This may explain the successful use of weak PDE5-inhibitors, such as dipyridamole, in anti-platelet therapy. In conclusion, increased NO signalling or PDE5 inhibition are attractive ways of increasing cGMP-cAMP cross-talk selectively in platelets.
Alterations in the brain that contribute to the development of epilepsy, also called epileptogenesis, are not well understood, which makes it difficult to develop strategies for preventing epilepsy. Here we have studied the role of the CRE binding transcription factors, cyclic-AMP responsive element modulator (CREM) and inducible cyclic-AMP early repressor (ICER), in the development of epilepsy following pilocarpine induced status epilepticus (SE) in mice. Following SE, ICER mRNA and protein are increased in neurons. The increase in ICER, however, is not necessary for neuronal injury following SE as pilocarpine treatment induces equivalent neuronal injury in pyramidal neurons of wild type and CREM/ICER null mice. Following SE, the CREM/ICER null mice develop a more severe epileptic phenotype experiencing approximately threefold more frequent spontaneous seizures. Together these data suggest that the increase in ICER mRNA following SE may have a role in suppressing the severity of epilepsy.
Cyclic AMP (cAMP) inhibits the proliferation of several tumor cells. We previously reported an antiproliferative effect of PKA I-selective cAMP analogs (8-PIP-cAMP and 8-HA-cAMP) on two human cancer cell lines of different origin. 8-Cl-cAMP, another cAMP analog with known antiproliferative properties, has been investigated as a potential anticancer drug. Here, we compared the antiproliferative effect of 8-Cl-cAMP and the PKA I-selective cAMP analogs in three human cancer cell lines (ARO, NPA and WRO). 8-Cl-cAMP and the PKA I-selective cAMP analogs had similarly potent antiproliferative effects on the BRAF-positive ARO and NPA cells, but not on the BRAF-negative WRO cells, in which only 8-Cl-cAMP consistently inhibited cell growth. While treatment with the PKA I-selective cAMP analogs was associated with growth arrest, 8-Cl-cAMP induced apoptosis. To further investigate the actions of 8-Cl-cAMP and the PKA I-selective cAMP analogs, we analyzed their effects on signaling pathways involved in cell proliferation and apoptosis. Interestingly, the PKA I-selective cAMP analogs, but not 8-Cl-cAMP, inhibited ERK phosphorylation, whereas 8-Cl-cAMP alone induced a progressive phosphorylation of the p38 mitogen-activated protein kinase (MAPK), via activation of AMPK by its metabolite 8-Cl-adenosine. Importantly, the pro-apoptotic effect of 8-Cl-cAMP could be largely prevented by pharmacological inhibition of the p38 MAPK. Altogether, these data suggest that 8-Cl-cAMP and the PKA I-selective cAMP analogs, though of comparable antiproliferative potency, act through different mechanisms. PKA I-selective cAMP analogs induce growth arrest in cells carrying the BRAF oncogene, whereas 8-Cl-cAMP induce apoptosis, apparently through activation of the p38 MAPK pathway.
Mammalian cyclic GMP-AMP synthase (cGAS) and its homologue dinucleotide cyclase in Vibrio cholerae (VcDncV) produce cyclic dinucleotides (CDNs) that participate in the defense against viral infection. Recently, scores of new cGAS/DncV-like nucleotidyltransferases (CD-NTases) were discovered, which produce various CDNs and cyclic trinucleotides (CTNs) as second messengers. Here, we present the crystal structures of EcCdnD, a CD-NTase from Enterobacter cloacae that produces cyclic AMP-AMP-GMP, in its apo-form and in complex with ATP, ADP and AMPcPP, an ATP analogue. Despite the similar overall architecture, the protein shows significant structural variations from other CD-NTases. Adjacent to the donor substrate, another nucleotide is bound to the acceptor binding site by a non-productive mode. Isothermal titration calorimetry results also suggest the presence of two ATP binding sites. GTP alone does not bind to EcCdnD, which however binds to pppApG, a possible intermediate. The enzyme is active on ATP or a mixture of ATP and GTP, and the best metal cofactor is Mg2+. The conserved residues Asp69 and Asp71 are essential for catalysis, as indicated by the loss of activity in the mutants. Based on structural analysis and comparison with VcDncV and RNA polymerase, a tentative catalytic pathway for the CTN-producing EcCdnD is proposed.
Cyclic dimeric adenosine monophosphate (cyclic-di-AMP) is a nucleotide second messenger present in Gram-positive bacteria, Gram-negative bacteria and some Archaea. The intracellular concentration of cyclic-di-AMP is adjusted in response to environmental and cellular cues, primarily through the activities of synthesis and degradation enzymes. It performs its role by binding to protein and riboswitch receptors, many of which contribute to osmoregulation. Imbalances in cyclic-di-AMP can lead to pleiotropic phenotypes, affecting aspects such as growth, biofilm formation, virulence, and resistance to osmotic, acid, and antibiotic stressors. This review focuses on cyclic-di-AMP signalling in lactic acid bacteria (LAB) incorporating recent experimental discoveries and presenting a genomic analysis of signalling components from a variety of LAB, including those found in food, and commensal, probiotic, and pathogenic species. All LAB possess enzymes for the synthesis and degradation of cyclic-di-AMP, but are highly variable with regards to the receptors they possess. Studies in Lactococcus and Streptococcus have revealed a conserved function for cyclic-di-AMP in inhibiting the transport of potassium and glycine betaine, either through direct binding to transporters or to a transcriptional regulator. Structural analysis of several cyclic-di-AMP receptors from LAB has also provided insights into how this nucleotide exerts its influence.
The protozoan parasite Trypanosoma brucei engages in surface-induced social behavior, termed social motility, characterized by single cells assembling into multicellular groups that coordinate their movements in response to extracellular signals. Social motility requires sensing and responding to extracellular signals, but the underlying mechanisms are unknown. Here we report that T. brucei social motility depends on cyclic AMP (cAMP) signaling systems in the parasite's flagellum (synonymous with cilium). Pharmacological inhibition of cAMP-specific phosphodiesterase (PDE) completely blocks social motility without impacting the viability or motility of individual cells. Using a fluorescence resonance energy transfer (FRET)-based sensor to monitor cAMP dynamics in live cells, we demonstrate that this block in social motility correlates with an increase in intracellular cAMP levels. RNA interference (RNAi) knockdown of the flagellar PDEB1 phenocopies pharmacological PDE inhibition, demonstrating that PDEB1 is required for social motility. Using parasites expressing distinct fluorescent proteins to monitor individuals in a genetically heterogeneous community, we found that the social motility defect of PDEB1 knockdowns is complemented by wild-type parasites in trans. Therefore, PDEB1 knockdown cells are competent for social motility but appear to lack a necessary factor that can be provided by wild-type cells. The combined data demonstrate that the role of cyclic nucleotides in regulating microbial social behavior extends to African trypanosomes and provide an example of transcomplementation in parasitic protozoa.
Biofilm-related infections are a major contributor to human disease, and the capacity for surface attachment and biofilm formation are key attributes for the pathogenesis of microbes. Serratia marcescens type I fimbriae-dependent biofilms are coordinated by the adenylate cyclase, CyaA, and the cyclic 3',5'-adenosine monophosphate (cAMP)-cAMP receptor protein (CRP) complex. This study uses S. marcescens as a model system to test the role of cAMP-phosphodiesterase activity in controlling biofilm formation. Herein we describe the characterization of a putative S. marcescens cAMP-phosphodiesterase gene (SMA3506), designated as cpdS, and demonstrated to be a functional cAMP-phosphodiesterase both in vitro and in vivo. Deletion of cpdS resulted in defective biofilm formation and reduced type I fimbriae production, whereas multicopy expression of cpdS conferred a type I fimbriae-dependent hyper-biofilm. Together, these results support a model in which bacterial cAMP-phosphodiesterase activity modulates biofilm formation.
1. The aim of this study was to determine the conditions under which the alpha2-adrenoceptor agonist UK14304 produces vasoconstriction in the porcine isolated ear artery. 2. UK14304 (0.3 microM) produced a small contraction of porcine isolated ear arteries which was 7.8+/-3.3% of the response to 60 mM KCl. Similar sized contractions were obtained after precontraction with either 30 nM angiotensin II, or 0.1 microM U46619 (8.2+/-1.8% and 10.2+/-2.6% of 60 mM KCl response, respectively). However, an enhanced alpha2-adrenoceptor response was uncovered if the tissue was precontracted with U46619, and relaxed back to baseline with 1-2 microM forskolin before the addition of UK14304 (46.9+/-9.6% of 60 mM KCl response). 3. The enhanced responses to UK14304 in the presence of U46619 and forskolin were not inhibited by the alpha1-adrenoceptor antagonist prazosin (0.1 microM), but were inhibited by the alpha2-adrenoceptor antagonist rauwolscine (1 microM), indicating that the enhanced responses were mediated via postjunctional alpha2-adrenoceptors. 4. In the presence of 0.1 microM U46619 and 1 mM isobutylmethylxanthine (IBMX), 1 microM forskolin produced an increase in [3H]-cyclic AMP levels in porcine isolated ear arteries. Addition of 0.3 microM UK14304 prevented this increase. 5. The enhanced UK14304 response was dependent upon the agent used to relax the tissue. After relaxation of ear arteries precontracted with 10 nM U46619 and relaxed with forskolin the UK14304 response was 46.9+/-9.6% of the 60 mM KCl response, and after relaxation with sodium nitroprusside (SNP) the response was 24.8+3.3%. However, after relaxation of the tissue with levcromakalim the UK14304 response was only 8.2+/-1.7%, which was not different from the control response in the same tissues (12.2+/-5.6%). An enhanced contraction was also obtained after relaxation of the tissue with the cyclic AMP analogue dibutyryl cyclic AMP (23.2+/-1.3%) indicating that at least part of the enhanced response to UK14304 is independent of the ability of the agonist to inhibit cyclic AMP production. 6. Relaxation of U46619 contracted ear arteries with SNP could be inhibited by the NO-sensitive guanylyl-cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) indicating that production of cyclic GMP is necessary for the relaxant effect of SNP. However, ODQ had no effect on the relaxation of tissue by forskolin, suggesting that this compound does not act via production of cyclic GMP. Biochemical studies showed that while forskolin increases the levels of cyclic AMP in the tissues, SNP had no effect on the levels of this cyclic nucleotide. 7. In conclusion, enhanced contractions to the alpha2-adrenoceptor agonist UK14304 can be uncovered in porcine isolated ear arteries by precontracting the tissue with U46619, followed by relaxation back to baseline with forskolin, SNP or dibutyryl cyclic AMP before addition of UK14304. There was a greater contractile response to UK14304 after relaxation with forskolin than with SNP or dibutyryl cyclic AMP, suggesting that cyclic AMP-dependent and- independent mechanisms are involved in the enhancement of the UK14304 response.
1. The objective of the present paper was to evaluate the relevance of neuronal balance of cyclic AMP and cyclic GMP concentration for functional regulation of nociceptor sensitivity during inflammation. 2. Injection of PGE2 (10-100 ng paw-1) evoked a dose-dependent hyperalgesic effect which was mediated via a cyclic AMP-activated protein kinase (PKA) inasmuch as hyperalgesia was blocked by the PKA inhibitor H89. 3. The PDE4 inhibitor rolipram and RP73401, but not PDE3 and PDE5 inhibitors potentiated the hyperalgesic effects of PGE2. The hyperalgesic effect of dopamine was also enhanced by rolipram. Moreover, rolipram significantly potentiated hyperalgesia induced by carrageenan, bradykinin, TNF alpha, IL-1 beta, IL-6 and IL-8. This suggests that neuronal cyclic AMP mediates the prostanoid and sympathetic components of mechanical hyperalgesia. Moreover, in the neuron cyclic AMP is mainly metabolized by PDE4. 4. To examine the role of the NO/cyclic GMP pathway in modulating mechanical hyperalgesia, we tested the effects of the soluble guanylate cyclase inhibitor, ODQ. This substance counteracts the inhibitory effects of the NO donor, SNAP, on the hyperalgesia induced by PGE2. 5. The ODQ potentiated hyperalgesia induced by carrageenan, bradykinin, TNF alpha, IL-1 beta, IL-6 and IL-8. In contrast, ODQ had no significant effect on the hyperalgesia induced by PGE2 and dopamine. This indicates that the hyperalgesic cytokines may activate soluble guanylate cyclase, which down-regulate the ability of these substances to cause hyperalgesia. This event appears not to be mediated by prostaglandin or dopamine. 6. In conclusion, the results presented in this paper confirm an association between (i) hyperalgesia and elevated levels of cyclic AMP as well as (ii) antinociception and elevated levels of cyclic GMP. The intracellular levels of cyclic AMP that enhance hyperalgesia are controlled by the PDE4 isoform and appear to result in activation of protein kinase A whereas the intracellular levels of cyclic GMP results from activation of a soluble guanylate cyclase.
In this study we determine different signaling pathways involved in beta(3) adrenoceptor (beta(3)-AR) dependent frequency stimulation in isolated rodent atria. Promiscuous coupling between different G-proteins and beta(3)-AR could explain the multiple functional effects of beta(3)-AR stimulation. We examine the mechanisms and functional consequences of dual adenylate cyclase and guanylate cyclase pathways coupling to beta(3)-AR in isolated rodent atria. The beta(3)-AR selective agonists ZD 7114 and ICI 215001 stimulated in a dose-dependent manner the contraction frequency that significantly correlated with cyclic AMP (cAMP) accumulation. Inhibition of adenylate cyclase shifted the chronotropic effect to the right. On the other hand, the ZD 7114 activity on frequency was enhanced by the inhibition of nitric oxide synthase (NOS) and soluble guanylate cyclase. This countervailing negative chronotropic nitric oxide-cyclic GMP (NO-cGMP) significantly correlated with the increase on NOS activity and cGMP accumulation. Current analysis showed a negative cross talk between cAMP chronotropic and NO-cGMP effects by inhibition of phospholipase C (PLC), calcium/calmodulin (CaM), protein kinase C (PKC), NOS isoforms and Gi-protein on the effects of beta(3)-AR stimulation. RT-PCR detected both eNOS and nNOS in isolated rat atria. NOS isoforms performed independently. Only nNOS participated in limiting the effect of beta(3)-AR stimulation. In eNOS-KO (eNOS-/-) mice the chronotropic effect of beta(3)-AR agonists did not differ from wild type (WT) mice atria, but it was increased by the inhibition of nNOS activity. Our results suggest that the increase in frequency by beta(3)-AR activation on isolated rodent atria is associated to a parallel increases in cAMP. The nNOS-cGMP pathway negatively modulates beta(3)-AR activation. Multiple signal transduction pathways between G-protein and beta(3)-AR may protect myocardium from catecholamine-induced cardiotoxic effects.
The bacterial adenylyl cyclase toxins CyaA from Bordetella pertussis and edema factor from Bacillus anthracis as well as soluble guanylyl cyclase α(1)β(1) synthesize the cyclic pyrimidine nucleotide cCMP. These data raise the question to which effector proteins cCMP binds. Recently, we reported that cCMP activates the regulatory subunits RIα and RIIα of cAMP-dependent protein kinase. In this study, we used two cCMP agarose matrices as novel tools in combination with immunoblotting and mass spectrometry to identify cCMP-binding proteins. In agreement with our functional data, RIα and RIIα were identified as cCMP-binding proteins. These data corroborate the notion that cAMP-dependent protein kinase may serve as a cCMP target.
Cyclic adenosine monophosphate (cyclic AMP) is a second messenger, which is involved in the regulation of various cellular processes, including neuronal firing rate, synaptic plasticity, axon formation and axon elongation in brain. Although the main molecules in the cAMP-mediated signaling pathway are well studied, the spatio-temporal dynamics of the cAMP remain to be elucidated. Live imaging is an informative tool to investigate the cell signaling dynamics. It allows continuous monitoring of a specific cell over a period of time. Thus, optical probes for cAMP are important tools for studying the dynamics of cAMP signaling. Multiple genetically encoded cAMP probes are available [1], [2], including Förster resonance energy transfer (FRET) based or circular permutated fluorescent protein (cpFP) based probes. cpFP-based probes have an advantage of easier handling than FRET-based probes caused by monomeric detection and smaller molecular size. However, there is no cAMP probe compatible with violet light excitation. Therefore, we fused violet light excitable cpGFP to cyclic nucleotide binding domain (CBD) in E. coli cAMP receptor protein. This construct successfully responded to cAMP concentration changes. We show here the spectra data and live-cell imaging data of the violet light excitable cAMP probe which can be used for multi-signal fluorescence imaging.
Cyclic GMP-AMP synthase (cGAS, cGAMP synthase) plays crucial roles in autoimmune disease, anti-tumor response, anti-senescence and anti-inflammatory response. Many studies have focused on cGAS-mediated signaling pathway. However, transcriptional mechanisms of cGAS gene have remained largely unknown. Here, we cloned the cGAS promoter region and characterized the molecular mechanisms controlling the cGAS transcriptional activity. By a series of 5' deletion and promoter constructions, we showed that the region (-414 to +76 relatives to the transcription start site) was sufficient for promoter activity. Mutation of Sp1 and CREB binding sites in this promoter region led to an apparent reduction of the cGAS promoter activity. Overexpression of Sp1 and CREB could obviously enhance promoter activity, whereas knocking-down of endogenous Sp1 and CREB markedly restrained the cGAS promoter activity. Sp1 and CREB binding to the cGAS promoter region in vivo was verified by Chromatin immunoprecipitation assay. These results pointed out that transcription factors Sp1 and CREB regulate the transcription of the cGAS gene.
The mechanism of adenosine-induced inhibition of ganglionic transmission was investigated in the isolated superior cervical ganglion (SCG) of the rat. The inhibitory effect of adenosine on the postganglionic compound action potential (CAP) was antagonized by pretreatment of ganglia with forskolin, isoproterenol (IPNE), arginine vasopressin (AVP), or papaverine, all of which are known to increase tissue cAMP level by different mechanisms. Furthermore, pretreatment of ganglia with the adenylate cyclase inhibitor SQ 22, 536, or the phosphodiesterase activator imidazole reversed the effects of IPNE and forskolin. Pretreatment with 8-bromo-cAMP, resulted in a marked antagonism of the adenosine-induced inhibition. By themselves, none of these drugs had any significant effect on the CAP. Adenosine slightly but significantly decreased the basal level of cAMP in untreated ganglia. Formation of cAMP induced by IPNE was markedly reduced by adenosine. This was largely reversed in the presence of the adenosine A1 receptor antagonist 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX) but not the A2 receptor antagonist 3, 7-dimethyl-1-propargylxanthine (DPMX). We conclude that the inhibition of ganglionic transmission by adenosine involves reduction of cAMP formation through activation of A1 receptors.
Waves of spontaneous activity sweep across the developing mammalian retina and influence the pattern of central connections made by ganglion cell axons. These waves are driven by synaptic input from amacrine cells. We show that cholinergic synaptic transmission during waves is not blocked by TTX, indicating that release from starburst amacrine cells is independent of sodium action potentials. The spatiotemporal properties of the waves are regulated by endogenous release of adenosine, which sets intracellular cAMP levels through activation of A2 receptors present on developing amacrine and ganglion cells. Increasing cAMP levels increase the size, speed, and frequency of the waves. Conversely, inhibiting adenylate cyclase or PKA prevents wave activity. Together, these results imply a novel mechanism in which levels of cAMP within an immature retinal circuit regulate the precise spatial and temporal patterns of spontaneous neural activity.
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