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Sample records for adenylyl cyclase signaling

  1. Calmodulin-regulated adenylyl cyclases and neuromodulation.

    PubMed

    Xia, Z; Storm, D R

    1997-06-01

    Coincidence detection and crosstalk between signal transduction systems play very important regulatory roles in the nervous system, particularly in the regulation of transcription. Coupling of the Ca2+ and cAMP regulatory systems by calmodulin-regulated adenylyl cyclases is hypothesized to be important for some forms of synaptic plasticity, neuroendocrine function, and olfactory detection. Recent studies of a mutant mouse deficient in type I calmodulin-sensitive adenylyl cyclase have provided the first evidence that adenylyl cyclases are important for synaptic plasticity, as well as for learning and memory in vertebrates.

  2. AN ADENYLYL CYCLASE SIGNALING PATHWAY PREDICTS DIRECT DOPAMINERGIC INPUT TO VESTIBULAR HAIR CELLS

    PubMed Central

    DRESCHER, M. J.; CHO, W. J.; FOLBE, A. J.; SELVAKUMAR, D.; KEWSON, D. T.; ABU-HAMDAN, M. D.; OH, C. K.; RAMAKRISHNAN, N. A.; HATFIELD, J. S.; KHAN, K. M.; ANNE, S.; HARPOOL, E. C.; DRESCHER, D. G.

    2010-01-01

    Adenylyl cyclase signaling pathways have been identified in a model hair cell preparation from the trout saccule, for which the hair cell is the only intact cell type. The use of degenerate primers targeting cDNA sequence conserved across adenylyl cyclase (AC) isoforms, and RT-PCR, coupled with cloning of amplification products, indicated expression of AC9, AC7 and AC5/6, with cloning efficiencies of 11:5:2. AC9 and AC5/6 are inhibited by Ca2+, the former in conjunction with calcineurin, and message for calcineurin has also been identified in the trout saccular hair cell layer. AC7 is independent of Ca2+. Given the lack of detection of calcium/calmodulin-activated isoforms previously suggested to mediate adenylyl cyclase activation in the absence of Gαs in mammalian cochlear hair cells, the issue of hair-cell Gαs mRNA expression was re-examined in the teleost vestibular hair cell model. Two full-length coding sequences were obtained for Gαs/olf in the vestibular type II-like hair cells of the trout saccule. Two messages for Gαi have also been detected in the hair cell layer, one with homology to Gαi1 and the second with homology to Gαi3 of higher vertebrates. Both Gαs/olf protein and Gαi1/Gαi3 protein were immunolocalized to stereocilia and to the base of the hair cell, the latter consistent with sites of efferent input. While a signaling event coupling to Gαs/olf and Gαi1/Gαi3 in the stereocilia is currently unknown, signaling with Gαs/olf, Gαi3, and AC5/6 at the base of the hair cell would be consistent with transduction pathways activated by dopaminergic efferent input. mRNA for dopamine receptors D1A4 and five forms of dopamine D2 were found to be expressed in the teleost saccular hair cell layer, representing information on vestibular hair cell expression not directly available for higher vertebrates. Dopamine D1A receptor would couple to Gαolf and activation of AC5/6. Co-expression with dopamine D2 receptor, which itself couples to Gαi3 and AC

  3. Adenylyl cyclases in the digestive system.

    PubMed

    Sabbatini, Maria Eugenia; Gorelick, Fred; Glaser, Shannon

    2014-06-01

    Adenylyl cyclases (ACs) are a group of widely distributed enzymes whose functions are very diverse. There are nine known transmembrane AC isoforms activated by Gαs. Each has its own pattern of expression in the digestive system and differential regulation of function by Ca(2+) and other intracellular signals. In addition to the transmembrane isoforms, one AC is soluble and exhibits distinct regulation. In this review, the basic structure, regulation and physiological roles of ACs in the digestive system are discussed. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Adenylyl cyclases in the digestive system

    PubMed Central

    Sabbatini, Maria Eugenia; Gorelick, Fred; Glaser, Shannon

    2015-01-01

    Adenylyl cyclases (ACs) are a group of widely distributed enzymes whose functions are very diverse. There are nine known transmembrane AC isoforms activated by Gαs. Each has its own pattern of expression in the digestive system and differential regulation of function by Ca2+ and other intracellular signals. In addition to the transmembrane isoforms, one AC is soluble and exhibits distinct regulation. In this review, the basic structure, regulation and physiological roles of ACs in the digestive system are discussed. PMID:24521753

  5. An adenylyl cyclase signaling pathway predicts direct dopaminergic input to vestibular hair cells.

    PubMed

    Drescher, M J; Cho, W J; Folbe, A J; Selvakumar, D; Kewson, D T; Abu-Hamdan, M D; Oh, C K; Ramakrishnan, N A; Hatfield, J S; Khan, K M; Anne, S; Harpool, E C; Drescher, D G

    2010-12-29

    Adenylyl cyclase (AC) signaling pathways have been identified in a model hair cell preparation from the trout saccule, for which the hair cell is the only intact cell type. The use of degenerate primers targeting cDNA sequence conserved across AC isoforms, and reverse transcription-polymerase chain reaction (RT-PCR), coupled with cloning of amplification products, indicated expression of AC9, AC7 and AC5/6, with cloning efficiencies of 11:5:2. AC9 and AC5/6 are inhibited by Ca(2+), the former in conjunction with calcineurin, and message for calcineurin has also been identified in the trout saccular hair cell layer. AC7 is independent of Ca(2+). Given the lack of detection of calcium/calmodulin-activated isoforms previously suggested to mediate AC activation in the absence of Gαs in mammalian cochlear hair cells, the issue of hair-cell Gαs mRNA expression was re-examined in the teleost vestibular hair cell model. Two full-length coding sequences were obtained for Gαs/olf in the vestibular type II-like hair cells of the trout saccule. Two messages for Gαi have also been detected in the hair cell layer, one with homology to Gαi1 and the second with homology to Gαi3 of higher vertebrates. Both Gαs/olf protein and Gαi1/Gαi3 protein were immunolocalized to stereocilia and to the base of the hair cell, the latter consistent with sites of efferent input. Although a signaling event coupling to Gαs/olf and Gαi1/Gαi3 in the stereocilia is currently unknown, signaling with Gαs/olf, Gαi3, and AC5/6 at the base of the hair cell would be consistent with transduction pathways activated by dopaminergic efferent input. mRNA for dopamine receptors D1A4 and five forms of dopamine D2 were found to be expressed in the teleost saccular hair cell layer, representing information on vestibular hair cell expression not directly available for higher vertebrates. Dopamine D1A receptor would couple to Gαolf and activation of AC5/6. Co-expression with dopamine D2 receptor, which

  6. The "soluble" adenylyl cyclase in sperm mediates multiple signaling events required for fertilization.

    PubMed

    Hess, Kenneth C; Jones, Brian H; Marquez, Becky; Chen, Yanqiu; Ord, Teri S; Kamenetsky, Margarita; Miyamoto, Catarina; Zippin, Jonathan H; Kopf, Gregory S; Suarez, Susan S; Levin, Lonny R; Williams, Carmen J; Buck, Jochen; Moss, Stuart B

    2005-08-01

    Mammalian fertilization is dependent upon a series of bicarbonate-induced, cAMP-dependent processes sperm undergo as they "capacitate," i.e., acquire the ability to fertilize eggs. Male mice lacking the bicarbonate- and calcium-responsive soluble adenylyl cyclase (sAC), the predominant source of cAMP in male germ cells, are infertile, as the sperm are immotile. Membrane-permeable cAMP analogs are reported to rescue the motility defect, but we now show that these "rescued" null sperm were not hyperactive, displayed flagellar angulation, and remained unable to fertilize eggs in vitro. These deficits uncover a requirement for sAC during spermatogenesis and/or epididymal maturation and reveal limitations inherent in studying sAC function using knockout mice. To circumvent this restriction, we identified a specific sAC inhibitor that allowed temporal control over sAC activity. This inhibitor revealed that capacitation is defined by separable events: induction of protein tyrosine phosphorylation and motility are sAC dependent while acrosomal exocytosis is not dependent on sAC.

  7. Hypoxia induces cancer-associated cAMP/PKA signalling through HIF-mediated transcriptional control of adenylyl cyclases VI and VII.

    PubMed

    Simko, Veronika; Iuliano, Filippo; Sevcikova, Andrea; Labudova, Martina; Barathova, Monika; Radvak, Peter; Pastorekova, Silvia; Pastorek, Jaromir; Csaderova, Lucia

    2017-08-31

    Hypoxia is a phenomenon often arising in solid tumours, linked to aggressive malignancy, bad prognosis and resistance to therapy. Hypoxia-inducible factor-1 has been identified as a key mediator of cell and tissue adaptation to hypoxic conditions through transcriptional activation of many genes involved in glucose metabolism and other cancer-related processes, such as angiogenesis, cell survival and cell invasion. Cyclic adenosine 3'5'-monophosphate is one of the most ancient and evolutionarily conserved signalling molecules and the cAMP/PKA signalling pathway plays an important role in cellular adaptation to hypoxia. We have investigated possible new mechanisms behind hypoxic activation of the cAMP/PKA pathway. For the first time, we have shown that hypoxia induces transcriptional up-regulation of the system of adenylyl cyclases, enzymes responsible for cAMP production, in a panel of carcinoma cell lines of various origin. Our data prove functional relevance of the hypoxic increase of adenylyl cyclases VI and VII at least partially mediated by HIF-1 transcription factor. We have identified adenylyl cyclase VI and VII isoforms as mediators of cellular response to hypoxia, which led to the elevation of cAMP levels and enhanced PKA activity, with an impact on cell migration and pH regulation.

  8. The Type 3 Adenylyl Cyclase is Required for Novel Object Learning and Extinction of Contextual Memory: Role of cAMP Signaling in Primary Cilia

    PubMed Central

    Wang, Zhenshan; Phan, Trongha; Storm, Daniel R.

    2011-01-01

    Although primary cilia are found on neurons throughout the brain, their physiological function remains elusive. Human ciliopathies are associated with cognition defects and transgenic mice lacking proteins expressed in primary cilia exhibit defects in learning and memory. Recently, it was reported that mice lacking the G-protein coupling receptor somatostatin receptor-3 (SSTR3), a protein expressed predominately in the primary cilia of neurons, have defective memory for novel object recognition and lower cAMP levels in the brain. Since SSTR3 is coupled to regulation of adenylyl cyclase this suggests that adenylyl cyclase activity in primary cilia of CNS neurons may be critical for some forms of learning and memory. Because the type 3 adenylyl cyclase (AC3) is expressed in primary cilia of hippocampal neurons, we examined AC3−/− mice for several forms of learning and memory. Here, we report that AC3−/− mice show no short-term memory for novel objects and fail to exhibit extinction of contextual fear conditioning. They also show impaired learning and memory for temporally dissociated passive avoidance (TDPA). Since AC3 is exclusively expressed in primary cilia we conclude that cAMP signals generated within primary cilia contribute to some forms of learning and memory including extinction of contextual fear conditioning. PMID:21490195

  9. The type 3 adenylyl cyclase is required for novel object learning and extinction of contextual memory: role of cAMP signaling in primary cilia.

    PubMed

    Wang, Zhenshan; Phan, Trongha; Storm, Daniel R

    2011-04-13

    Although primary cilia are found on neurons throughout the brain, their physiological function remains elusive. Human ciliopathies are associated with cognition defects, and transgenic mice lacking proteins expressed in primary cilia exhibit defects in learning and memory. Recently, it was reported that mice lacking the G-protein-coupling receptor somatostatin receptor-3 (SSTR3), a protein expressed predominately in the primary cilia of neurons, have defective memory for novel object recognition and lower cAMP levels in the brain. Since SSTR3 is coupled to regulation of adenylyl cyclase, this suggests that adenylyl cyclase activity in primary cilia of CNS neurons may be critical for some forms of learning and memory. Because the type 3 adenylyl cyclase (AC3) is expressed in primary cilia of hippocampal neurons, we examined AC3(-/-) mice for several forms of learning and memory. Here, we report that AC3(-/-) mice show no short-term memory for novel objects and fail to exhibit extinction of contextual fear conditioning. They also show impaired learning and memory for temporally dissociative passive avoidance. Since AC3 is exclusively expressed in primary cilia, we conclude that cAMP signals generated within primary cilia contribute to some forms of learning and memory, including extinction of contextual fear conditioning.

  10. β3GnT2 Maintains Adenylyl Cyclase-3 Signaling and Axon Guidance Molecule Expression in the Olfactory Epithelium

    PubMed Central

    Faden, Ashley A.; Knott, Thomas K.

    2011-01-01

    In the olfactory epithelium (OE), odorant receptor stimulation generates cAMP signals that function in both odor detection and the regulation of axon guidance molecule expression. The enzyme that synthesizes cAMP, adenylyl cyclase 3 (AC3), is coexpressed in olfactory sensory neurons (OSNs) with poly-N-acetyllactosamine (PLN) oligosaccharides determined by the glycosyltransferase β3GnT2. The loss of either enzyme results in similar defects in olfactory bulb (OB) innervation and OSN survival, suggesting that glycosylation may be important for AC3 function. We show here that AC3 is extensively modified with N-linked PLN, which is essential for AC3 activity and localization. On Western blots, AC3 from the wild-type OE migrates diffusely as a heavily glycosylated 200 kDa band that interacts with the PLN-binding lectin LEA. AC3 from the β3GnT2−/− OE loses these PLN modifications, migrating instead as a 140 kDa glycoprotein. Furthermore, basal and forskolin-stimulated cAMP production is reduced 80–90% in the β3GnT2−/− OE. Although AC3 traffics normally to null OSN cilia, it is absent from axon projections that aberrantly target the OB. The cAMP-dependent guidance receptor neuropilin-1 is also lost from β3GnT2−/− OSNs and axons, while semaphorin-3A ligand expression is upregulated. In addition, kirrel2, a mosaically expressed adhesion molecule that functions in axon sorting, is absent from β3GnT2−/− OB projections. These results demonstrate that PLN glycans are essential in OSNs for proper AC3 localization and function. We propose that the loss of cAMP-dependent guidance cues is also a critical factor in the severe axon guidance defects observed in β3GnT2−/− mice. PMID:21525298

  11. Soluble Adenylyl Cyclase of Sea Urchin Spermatozoa

    PubMed Central

    Vacquier, Victor D.; Loza-Huerta, Arlet; García-Rincón, Juan; Darszon, Alberto; Beltrán, Carmen

    2014-01-01

    Fertilization, a key step in sexual reproduction, requires orchestrated changes in cAMP concentrations. It is notable that spermatozoa (sperm) are amongst the cell types with extremely high adenylyl cyclase (AC) activity. As production and consumption of this second messenger need to be locally regulated, the discovery of soluble AC (sAC) has broadened our understanding of how such cells deal with these requirements. In addition, because sAC is directly regulated by HCO3- it is able to translate CO2/HCO3-/pH changes into cAMP levels. Fundamental sperm functions such as maturation, motility regulation and the acrosome reaction are influenced by cAMP; this is especially true for sperm of the sea urchin (SU), an organism that has been a model in the study of fertilization for more than 130 years. Here we summarize the discovery and properties of SU sperm sAC, and discuss its involvement in sperm physiology. PMID:25064590

  12. Adenylyl cyclase and G-proteins in Phytomonas.

    PubMed

    Farber, M D; Montagna, A E; Paveto, C; Dollet, M; Sanchex-Moreno, M; Osuna, A; Torres, H N; Flawia, M M

    1995-01-01

    Phytomonas sp. membranes have an adenylyl cyclase activity which is greater in the presence of Mn2+ than with Mg2+. The Mg2+ and Mn2+ activity ratio varies from one membrane preparation to another, suggesting that the adenylyl cyclase has a variable activation state. A[35S]GTP-gamma-S-binding activity with a Kd of 171 nM was detected in Phytomonas membranes. Incubation of these membranes with activated cholera or pertussis toxin and [adenylate 23P]NAD+ led to incorporation of radioactivity into bands of about 40-44 kDa. Crude membranes were electrophoresed on SDS-polyacrylamide gels and analyzed, by Western blotting, with the 9188 anti-alpha[s] antibody and the AS/7 antibody (anti-alpha[i], anti-alpha[i1], and anti-alpha[i2]. These procedures resulted in the identification of polypeptides of approximately 40-44 kDa. Phytomonas adenylyl cyclase could be activated by treatment of membrane preparations with cholera toxin, in the presence of NAD+, while similar treatment with pertussis toxin did not affect this enzyme activity. These studies indicate that in Phytomonas, adenylyl cyclase activity is coupled to an unknown receptor entity through G alpha[s] proteins.

  13. Blocking adenylyl cyclase inhibits olfactory generator currents induced by "IP(3)-odors".

    PubMed

    Chen, S; Lane, A P; Bock, R; Leinders-Zufall, T; Zufall, F

    2000-07-01

    Vertebrate olfactory receptor neurons (ORNs) transduce odor stimuli into electrical signals by means of an adenylyl cyclase/cAMP second messenger cascade, but it remains widely debated whether this cAMP cascade mediates transduction for all odorants or only certain odor classes. To address this problem, we have analyzed the generator currents induced by odors that failed to produce cAMP in previous biochemical assays but instead produced IP(3) ("IP(3)-odors"). We show that in single salamander ORNs, sensory responses to "cAMP-odors" and IP(3)-odors are not mutually exclusive but coexist in the same cells. The currents induced by IP(3)-odors exhibit identical biophysical properties as those induced by cAMP odors or direct activation of the cAMP cascade. By disrupting adenylyl cyclase to block cAMP formation using two potent antagonists of adenylyl cyclase, SQ22536 and MDL12330A, we show that this molecular step is necessary for the transduction of both odor classes. To assess whether these results are also applicable to mammals, we examine the electrophysiological responses to IP(3)-odors in intact mouse main olfactory epithelium (MOE) by recording field potentials. The results show that inhibition of adenylyl cyclase prevents EOG responses to both odor classes in mouse MOE, even when "hot spots" with heightened sensitivity to IP(3)-odors are examined.

  14. Cyanobacteriochrome-based photoswitchable adenylyl cyclases (cPACs) for broad spectrum light regulation of cAMP levels in cells.

    PubMed

    Blain-Hartung, Matthew; Rockwell, Nathan C; Moreno, Marcus V; Martin, Shelley S; Gan, Fei; Bryant, Donald A; Lagarias, J Clark

    2018-06-01

    Class III adenylyl cyclases generate the ubiquitous second messenger cAMP from ATP often in response to environmental or cellular cues. During evolution, soluble adenylyl cyclase catalytic domains have been repeatedly juxtaposed with signal-input domains to place cAMP synthesis under the control of a wide variety of these environmental and endogenous signals. Adenylyl cyclases with light-sensing domains have proliferated in photosynthetic species depending on light as an energy source, yet are also widespread in nonphotosynthetic species. Among such naturally occurring light sensors, several flavin-based photoactivated adenylyl cyclases (PACs) have been adopted as optogenetic tools to manipulate cellular processes with blue light. In this report, we report the discovery of a cyanobacteriochrome-based photoswitchable adenylyl cyclase (cPAC) from the cyanobacterium Microcoleus sp. PCC 7113. Unlike flavin-dependent PACs, which must thermally decay to be deactivated, cPAC exhibits a bistable photocycle whose adenylyl cyclase could be reversibly activated and inactivated by blue and green light, respectively. Through domain exchange experiments, we also document the ability to extend the wavelength-sensing specificity of cPAC into the near IR. In summary, our work has uncovered a cyanobacteriochrome-based adenylyl cyclase that holds great potential for the design of bistable photoswitchable adenylyl cyclases to fine-tune cAMP-regulated processes in cells, tissues, and whole organisms with light across the visible spectrum and into the near IR.

  15. Pituitary adenylyl cyclase-activating polypeptide (PACAP) and its receptor (PAC1-R) are positioned to modulate afferent signaling in the cochlea.

    PubMed

    Drescher, M J; Drescher, D G; Khan, K M; Hatfield, J S; Ramakrishnan, N A; Abu-Hamdan, M D; Lemonnier, L A

    2006-09-29

    Pituitary adenylyl cyclase-activating polypeptide (PACAP), via its specific receptor pituitary adenylyl cyclase-activating polypeptide receptor 1 (PAC1-R), is known to have roles in neuromodulation and neuroprotection associated with glutamatergic and cholinergic neurotransmission, which, respectively, are believed to form the primary basis for afferent and efferent signaling in the organ of Corti. Previously, we identified transcripts for PACAP preprotein and multiple splice variants of its receptor, PAC1-R, in microdissected cochlear subfractions. In the present work, neural localizations of PACAP and PAC1-R within the organ of Corti and spiral ganglion were examined, defining sites of PACAP action. Immunolocalization of PACAP and PAC1-R in the organ of Corti and spiral ganglion was compared with immunolocalization of choline acetyltransferase (ChAT) and synaptophysin as efferent neuronal markers, and glutamate receptor 2/3 (GluR2/3) and neurofilament 200 as afferent neuronal markers, for each of the three cochlear turns. Brightfield microscopy giving morphological detail for individual immunolocalizations was followed by immunofluorescence detection of co-localizations. PACAP was found to be co-localized with ChAT in nerve fibers of the intraganglionic spiral bundle and beneath the inner and outer hair cells within the organ of Corti. Further, evidence was obtained that PACAP is expressed in type I afferent axons leaving the spiral ganglion en route to the auditory nerve, potentially serving as a neuromodulator in axonal terminals. In contrast to the efferent localization of PACAP within the organ of Corti, PAC1-R immunoreactivity was co-localized with afferent dendritic neuronal marker GluR2/3 in nerve fibers passing beneath and lateral to the inner hair cell and in fibers at supranuclear and basal sites on outer hair cells. Given the known association of PACAP with catecholaminergic neurotransmission in sympathoadrenal function, we also re-examined the issue

  16. Ocean acidification stimulates alkali signal pathway: A bicarbonate sensing soluble adenylyl cyclase from oyster Crassostrea gigas mediates physiological changes induced by CO2 exposure.

    PubMed

    Wang, Xiudan; Wang, Mengqiang; Jia, Zhihao; Wang, Hao; Jiang, Shuai; Chen, Hao; Wang, Lingling; Song, Linsheng

    2016-12-01

    Ocean acidification (OA) has been demonstrated to have severe effects on marine organisms, especially marine calcifiers. However, the impacts of OA on the physiology of marine calcifiers and the underlying mechanisms remain unclear. Soluble adenylyl cyclase (sAC) is an acid-base sensor in response to [HCO 3 - ] and an intracellular source of cyclic AMP (cAMP). In the present study, an ortholog of sAC was identified from pacific oyster Crassostrea gigas (designated as CgsAC) and the catalytic region of CgsAC was cloned and expressed. Similar to the native CgsAC from gill tissues, the recombinant CgsAC protein (rCgsAC) exhibited [HCO 3 - ] mediated cAMP-forming activity, which could be inhibited by a small molecule KH7. After 16days of CO 2 exposure (pH=7.50), the mRNA transcripts of CgsAC increased in muscle, mantle, hepatopancreas, gill, male gonad and haemocytes, and two truncated CgsAC forms of 45kD and 20kD were produced. Cytosolic CgsAC could be translocated from the cytoplasm and nuclei to the membrane in response to CO 2 exposure. Besides, CO 2 exposure could increase the production of cAMP and intracellular pH of haemocytes, which was regulated by CgsAC (p<0.05), suggesting the existence of a [HCO 3 - ]/CgsAC/cAMP signal pathway in oyster. The elevated CO 2 could induce an increase of ROS level (p<0.05) and a decrease of phagocytic rate of haemocytes (p<0.05), which could be inhibited by KH7. The results collectively suggest that CgsAC is an important acid-base sensor in oyster and the [HCO 3 - ]/CgsAC/cAMP signal pathway might be responsible for intracellular alkalization effects on oxidative phosphorylation and innate immunity under CO 2 exposure. The changes of intracellular pH, ROS, and phagocytosis mediated by CgsAC might help us to further understand the effects of ocean acidification on marine calcifiers. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Adenylyl cyclase G is activated by an intramolecular osmosensor.

    PubMed

    Saran, Shweta; Schaap, Pauline

    2004-03-01

    Adenylyl cyclase G (ACG) is activated by high osmolality and mediates inhibition of spore germination by this stress factor. The catalytic domains of all eukaryote cyclases are active as dimers and dimerization often mediates activation. To investigate the role of dimerization in ACG activation, we coexpressed ACG with an ACG construct that lacked the catalytic domain (ACGDeltacat) and was driven by a UV-inducible promoter. After UV induction of ACGDeltacat, cAMP production by ACG was strongly inhibited, but osmostimulation was not reduced. Size fractionation of native ACG showed that dimers were formed between ACG molecules and between ACG and ACGDeltacat. However, high osmolality did not alter the dimer/monomer ratio. This indicates that ACG activity requires dimerization via a region outside the catalytic domain but that dimer formation does not mediate activation by high osmolality. To establish whether ACG required auxiliary sensors for osmostimulation, we expressed ACG cDNA in a yeast adenylyl cyclase null mutant. In yeast, cAMP production by ACG was similarly activated by high osmolality as in Dictyostelium. This strongly suggests that the ACG osmosensor is intramolecular, which would define ACG as the first characterized primary osmosensor in eukaryotes.

  18. Ca2+ -stimulated adenylyl cyclases regulate ERK-dependent activation of MSK1 during fear conditioning.

    PubMed

    Sindreu, Carlos Balet; Scheiner, Zachary S; Storm, Daniel R

    2007-01-04

    The cAMP and ERK/MAP kinase (MAPK) signal transduction pathways are critical for hippocampus-dependent memory, a process that depends on CREB-mediated transcription. However, the extent of crosstalk between these pathways and the downstream CREB kinase activated during memory formation has not been elucidated. Here we report that PKA, MAPK, and MSK1, a CREB kinase, are coactivated in a subset of hippocampal CA1 pyramidal neurons following contextual fear conditioning. Activation of PKA, MAPK, MSK1, and CREB is absolutely dependent on Ca(2+)-stimulated adenylyl cyclase activity. We conclude that adenylyl cyclase activity supports the activation of MAPK, and that MSK1 is the major CREB kinase activated during training for contextual memory.

  19. Ca2+-Stimulated Adenylyl Cyclases Regulate ERK-Dependent Activation of MSK1 During Fear Conditioning

    PubMed Central

    Sindreu, Carlos Balet; Scheiner, Zachary S.; Storm, Daniel R.

    2007-01-01

    The cAMP and ERK/MAP kinase (MAPK) signal transduction pathways are critical for hippocampus-dependent memory, a process that depends on CREB-mediated transcription. However, the extent of crosstalk between these pathways and the downstream CREB kinase activated during memory formation have not been elucidated. Here we report that PKA, MAPK, and MSK1, a CREB kinase, are co-activated in a subset of hippocampal CA1 pyramidal neurons following contextual fear conditioning. Activation of PKA, MAPK, MSK1, and CREB is absolutely dependent on Ca2+-stimulated adenylyl cyclase activity. We conclude that adenylyl cyclase activity supports the activation of MAPK, and that MSK1 is the major CREB kinase activated during training for contextual memory. PMID:17196532

  20. Cloning and expression of a Ca(2+)-inhibitable adenylyl cyclase from NCB-20 cells.

    PubMed Central

    Yoshimura, M; Cooper, D M

    1992-01-01

    A cDNA that encodes an adenylyl cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] has been cloned from NCB-20 cells, in which adenylyl cyclase activity is inhibited by Ca2+ at physiological concentrations. The cDNA clone (5.8 kilobases) was isolated by polymerase chain reaction (PCR) using degenerate primers designed by comparison of three adenylyl cyclase sequences (types I, II, and III) and subsequent library screening. Northern analysis revealed expression of mRNA (6.1 kilobases) corresponding to this cDNA in cardiac tissue, which is a prominent source of Ca(2+)-inhibitable adenylyl cyclase. The clone encodes a protein of 1165 amino acids, whose hydrophilicity profile was very similar to those of other mammalian adenylyl cyclases that have recently been cloned. A noticeable difference between this protein and other adenylyl cyclases was a lengthy aminoterminal region before the first transmembrane span. Transient expression of this cDNA in the human embryonic kidney cell line 293 revealed a 3-fold increase in cAMP production in response to forskolin compared with control transfected cells. In purified plasma membranes from transfected cells, increased adenylyl cyclase activity was also detected, which was susceptible to inhibition by submicromolar Ca2+. Thus, this adenylyl cyclase seems to represent the Ca(2+)-inhibitable form that is encountered in NCB-20 cells, cardiac tissue, and elsewhere. Its identification should permit a determination of the structural features that determine the mode of regulation of adenylyl cyclase by Ca2+. Images PMID:1379717

  1. A role for calmodulin-stimulated adenylyl cyclases in cocaine sensitization.

    PubMed

    DiRocco, Derek P; Scheiner, Zachary S; Sindreu, Carlos Balet; Chan, Guy C-K; Storm, Daniel R

    2009-02-25

    Cocaine sensitization is produced by repeated exposure to the drug and is thought to reflect neuroadaptations that contribute to addiction. Here, we identify the Ca(2+)/calmodulin-stimulated adenylyl cyclases, type 1 (AC1) and type 8 (AC8), as novel regulators of this behavioral plasticity. We show that, whereas AC1 and AC8 single knock-out mice (AC1(-/-) and AC8(-/-)) exhibit Ca(2+)-stimulated adenylyl cyclase activity in striatal membrane fractions, AC1/8 double-knock-out (DKO) mice do not. Furthermore, DKO mice are acutely supersensitive to low doses of cocaine and fail to display locomotor sensitization after chronic cocaine treatment. Because of the known role for the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase signaling pathway in cocaine-induced behavioral plasticity and its coupling to calcium-stimulated cAMP signaling in the hippocampus, we measured phosphorylated ERK (pERK) levels in the striatum. Under basal conditions, pERK is upregulated in choline acetyltransferase-positive interneurons in DKO mice relative to wild-type (WT) controls. After acute cocaine treatment, pERK signaling is significantly suppressed in medium spiny neurons (MSNs) of DKO mice relative to WT mice. In addition to the lack of striatal ERK activation by acute cocaine, signaling machinery downstream of ERK is uncoupled in DKO mice. We demonstrate that AC1 and AC8 are necessary for the phosphorylation of mitogen and stress-activated kinase-1 (pMSK1) at Ser376 and Thr581 and cAMP response element-binding protein (pCREB) at Ser133 after acute cocaine treatment. Our results demonstrate that the Ca(2+)-stimulated adenylyl cyclases regulate long-lasting cocaine-induced behavioral plasticity via activation of the ERK/MSK1/CREB signaling pathway in striatonigral MSNs.

  2. A Role for Calmodulin-Stimulated Adenylyl Cyclases in Cocaine Sensitization

    PubMed Central

    DiRocco, Derek P.; Scheiner, Zachary S.; Sindreu, Carlos Balet; Chan, Guy C-K; Storm, Daniel R.

    2009-01-01

    Cocaine sensitization is produced by repeated exposure to the drug and is thought to reflect neuroadaptations that contribute to addiction. Here, we identify the Ca2+/calmodulin-stimulated adenylyl cyclases, type 1 (AC1) and type 8 (AC8), as novel regulators of this behavioral plasticity. We show that while AC1 and AC8 single knockout mice (AC1−/− and AC8−/−) exhibit Ca2+-stimulated adenylyl cyclase activity in striatal membrane fractions, AC1/8 double-knockout (DKO) mice do not. Furthermore, DKO mice are acutely supersensitive to low doses of cocaine and fail to display locomotor sensitization following chronic cocaine treatment. Because of the known role for the ERK/MAP kinase signaling pathway in cocaine-induced behavioral plasticity and its coupling to calcium-stimulated cAMP signaling in the hippocampus, we measured phosphorylated extracellular signal-regulated kinase (pERK) levels in the striatum. Under basal conditions, pERK is upregulated in choline acetyltransferase positive (ChAT+) interneurons in DKO mice relative to wild-type (WT) controls. Following acute cocaine treatment, pERK signaling is significantly suppressed in medium spiny neurons (MSNs) of DKO mice relative to WT mice. In addition to the lack of striatal ERK activation by acute cocaine, signaling machinery downstream of ERK is uncoupled in DKO mice. We demonstrate that AC1 and AC8 are necessary for the phosphorylation of mitogen and stress-activated kinase-1 (pMSK1) at Ser376 and Thr581, and cAMP response element-binding protein (pCREB) at Ser133 following acute cocaine treatment. Our results demonstrate that the Ca2+-stimulated adenylyl cyclases regulate long-lasting cocaine-induced behavioral plasticity via activation of the ERK/MSK1/CREB signaling pathway in striatonigral MSNs. PMID:19244515

  3. The Diurnal Oscillation of MAP Kinase and Adenylyl Cyclase Activities in the Hippocampus Depends on the SCN

    PubMed Central

    Phan, Trongha; Chan, Guy; Sindreu, Carlos; Eckel-Mahan, Kristin; Storm, Daniel R.

    2011-01-01

    Consolidation of hippocampus dependent memory is dependent on activation of the cAMP/ Erk/MAPK signal transduction pathway in the hippocampus. Recently, we discovered that adenylyl cyclase and MAPK activities undergo a circadian oscillation in the hippocampus and that inhibition of this oscillation impairs contextual memory. This suggests the interesting possibility that the persistence of hippocampus-dependent memory depends upon the reactivation of MAPK in the hippocampus during the circadian cycle. A key unanswered question is whether the circadian oscillation of this signaling pathway is intrinsic to the hippocampus or is driven by the master circadian clock in the suprachiasmatic nucleus (SCN). To address this question, we ablated the SCN of mice by electrolytic lesion and examined hippocampus-dependent memory as well as adenylyl cyclase and MAPK activities. Electrolytic lesion of the SCN two days after training for contextual fear memory reduced contextual memory measured two weeks after training indicating that maintenance of contextual memory depends on the SCN. Spatial memory was also compromised in SCN-lesioned mice. Furthermore, the diurnal oscillation of adenylyl cyclase and MAPK activities in the hippocampus was destroyed by lesioning of the SCN. These data suggest that hippocampus-dependent long-term memory is dependent on the SCN-controlled oscillation of the adenylyl cyclase/MAPK pathway in the hippocampus. PMID:21775607

  4. Regulator of G-protein signaling 6 (RGS6) promotes anxiety and depression by attenuating serotonin-mediated activation of the 5-HT1A receptor-adenylyl cyclase axis

    PubMed Central

    Stewart, Adele; Maity, Biswanath; Wunsch, Amanda M.; Meng, Fantao; Wu, Qi; Wemmie, John A.; Fisher, Rory A.

    2014-01-01

    Targeting serotonin (5-HT) bioavailability with selective 5-HT reuptake inhibitors (SSRIs) remains the most widely used treatment for mood disorders. However, their limited efficacy, delayed onset of action, and side effects restrict their clinical utility. Endogenous regulator of G-protein signaling (RGS) proteins have been implicated as key inhibitors of 5-HT1ARs, whose activation is believed to underlie the beneficial effects of SSRIs, but the identity of the specific RGS proteins involved remains unknown. We identify RGS6 as the critical negative regulator of 5-HT1AR-dependent antidepressant actions. RGS6 is enriched in hippocampal and cortical neurons, 5-HT1AR-expressing cells implicated in mood disorders. RGS6−/− mice exhibit spontaneous anxiolytic and antidepressant behavior rapidly and completely reversibly by 5-HT1AR blockade. Effects of the SSRI fluvoxamine and 5-HT1AR agonist 8-OH-DPAT were also potentiated in RGS6+/− mice. The phenotype of RGS6−/− mice was associated with decreased CREB phosphorylation in the hippocampus and cortex, implicating enhanced Gαi-dependent adenylyl cyclase inhibition as a possible causative factor in the behavior observed in RGS6−/− animals. Our results demonstrate that by inhibiting serotonergic innervation of the cortical-limbic neuronal circuit, RGS6 exerts powerful anxiogenic and prodepressant actions. These findings indicate that RGS6 inhibition may represent a viable means to treat mood disorders or enhance the efficacy of serotonergic agents.—Stewart, A., Maity, B., Wunsch, A. M., Meng, F., Wu, Q., Wemmie, J. A., Fisher, R. A. Regulator of G-protein signaling 6 (RGS6) promotes anxiety and depression by attenuating serotonin-mediated activation of the 5-HT1A receptor-adenylyl cyclase axis. PMID:24421401

  5. Type 1 Adenylyl Cyclase is Essential for Maintenance of Remote Contextual Fear Memory

    PubMed Central

    Shan, Qiang; Chan, Guy C.-K.; Storm, Daniel R.

    2008-01-01

    Although molecular mechanisms for hippocampus-dependent memory have been extensively studied, much less is known about signaling events important for remote memory. Here we report that mice lacking type 1 adenylyl cyclase (AC1) are able to establish and retrieve remote contextual memory but unable to sustain it as long as wild type mice. Interestingly, mice over-expressing AC1 show superior remote contextual memory even though they exhibit normal hippocampus-dependent contextual memory. These data illustrate that calcium coupling to cAMP contributes to the stability of remote memory and identifies AC1 as a potential drug target site to improve long-term remote memory. PMID:19036980

  6. Effects of Forskolin on Kupffer Cell Production of Interleukin-10 and Tumor Necrosis Factor Alpha Differ from Those of Endogenous Adenylyl Cyclase Activators: Possible Role for Adenylyl Cyclase 9

    PubMed Central

    Dahle, Maria K.; Myhre, Anders E.; Aasen, Ansgar O.; Wang, Jacob E.

    2005-01-01

    Proinflammatory cytokines like tumor necrosis factor alpha (TNF-α) that are released from Kupffer cells may trigger liver inflammation and damage. Hence, endogenous mechanisms for limiting TNF-α expression are crucial for avoiding the development of sepsis. Such mechanisms include the anti-inflammatory actions of interleukin-10 (IL-10) as well as signaling induced by the intracellular second messenger cyclic AMP (cAMP). Kupffer cells express several receptors that activate cAMP synthesis, including E-prostanoid receptors and β-adrenergic receptors. The expression and role of specific adenylyl cyclases in the inhibition of Kupffer cell activation have so far not been subject to study. Pretreatment of rat Kupffer cell cultures with cAMP analogues [8-(4-chlorophenyl)-thio-cAMP], adenylyl cyclase activator (forskolin), or ligands for G-coupled receptors (isoproterenol or prostaglandin E2) 30 min before the addition of lipopolysaccharide (LPS) (1 μg/ml) caused attenuated TNF-α levels in culture medium (forskolin/isoproterenol, P ≤ 0.05; prostaglandin E2, P ≤ 0.01). Forskolin also reduced IL-10 mRNA and protein (P ≤ 0.05), which was not observed with the other cAMP-inducing agents. Furthermore, we found that rat Kupffer cells express high levels of the forskolin-insensitive adenylyl cyclase 9 compared to whole liver and that this expression is down-regulated by LPS (P ≤ 0.05). We conclude that regulation of TNF-α and IL-10 in Kupffer cells depends on the mechanism by which cAMP is elevated. Forskolin and prostaglandin E2 differ in their effects, which suggests a possible role of forskolin-insensitive adenylyl cyclases like adenylyl cyclase 9. PMID:16239525

  7. Adenylyl cyclase 3/adenylyl cyclase-associated protein 1 (CAP1) complex mediates the anti-migratory effect of forskolin in pancreatic cancer cells.

    PubMed

    Quinn, Sierra N; Graves, Sarai H; Dains-McGahee, Clayton; Friedman, Emilee M; Hassan, Humma; Witkowski, Piotr; Sabbatini, Maria E

    2017-04-01

    Pancreatic cancer is one of the most lethal human malignancies. A better understanding of the intracellular mechanism of migration and invasion is urgently needed to develop treatment that will suppress metastases and improve overall survival. Cyclic adenosine monophosphate (cyclic AMP) is a second messenger that has shown to regulate migration and invasion of pancreatic cancer cells. The rise of cyclic AMP suppressed migration and invasion of pancreatic ductal adenocarcinoma cells. Cyclic AMP is formed from cytosolic ATP by the enzyme adenylyl cyclase (AC). There are ten isoforms of ACs; nine are anchored in the plasma membrane and one is soluble. What remains unknown is the extent to which the expression of transmembrane AC isoforms is both modified in pancreatic cancer and mediates the inhibitory effect of forskolin on cell motility. Using real-time PCR analysis, ADCY3 was found to be highly expressed in pancreatic tumor tissues, resulting in a constitutive increase in cyclic AMP levels. On the other hand, ADCY2 was down-regulated. Migration, invasion, and filopodia formation in two different pancreatic adenocarcinoma cell lines, HPAC and PANC-1 deficient in AC1 or AC3, were studied. We found that AC3, upon stimulation with forskolin, enhanced cyclic AMP levels and inhibited cell migration and invasion. Unlikely to be due to a cytotoxic effect, the inhibitory effects of forskolin involved the quick formation of AC3/adenylyl cyclase-associated protein 1 (CAP1)/G-actin complex, which inhibited filopodia formation and cell motility. Using Western blotting analysis, forskolin, through AC3 activation, caused phosphorylation of CREB, but not ERK. The effect of CREB phosphorylation is likely to be associated with long-term signaling changes. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  8. Activation and inhibition of adenylyl cyclase isoforms by forskolin analogs.

    PubMed

    Pinto, Cibele; Papa, Dan; Hübner, Melanie; Mou, Tung-Chung; Lushington, Gerald H; Seifert, Roland

    2008-04-01

    Adenylyl cyclase (AC) isoforms 1 to 9 are differentially expressed in tissues and constitute an interesting drug target. ACs 1 to 8 are activated by the diterpene, forskolin (FS). It is unfortunate that there is a paucity of AC isoform-selective activators. To develop such compounds, an understanding of the structure/activity relationships of diterpenes is necessary. Therefore, we examined the effects of FS and nine FS analogs on ACs 1, 2, and 5 expressed in Spodoptera frugiperda insect cells. Diterpenes showed the highest potencies at AC1 and the lowest potencies at AC2. We identified full agonists, partial agonists, antagonists, and inverse agonists, i.e., diterpenes that reduced basal AC activity. Each AC isoform exhibited a distinct pharmacological profile. AC2 showed the highest basal activity of all AC isoforms and highest sensitivity to inverse agonistic effects of 1-deoxy-forskolin, 7-deacetyl-1,9-dideoxy-forskolin, and, particularly, BODIPY-forskolin. In contrast, BODIPY-forskolin acted as partial agonist at the other ACs. 1-Deoxy-forskolin analogs were devoid of agonistic activity at ACs but antagonized the effects of FS in a mixed competitive/noncompetitive manner. At purified catalytic AC subunits, BODIPY-forskolin acted as weak partial agonist/strong partial antagonist. Molecular modeling revealed that the BODIPY group rotates promiscuously outside of the FS-binding site. Collectively, ACs are not uniformly activated and inhibited by FS and FS analogs, demonstrating the feasibility to design isoform-selective FS analogs. The two- and multiple-state models, originally developed to conceptualize ligand effects at G-protein-coupled receptors, can be applied to ACs to explain certain experimental data.

  9. Functional characterization of transmembrane adenylyl cyclases from the honeybee brain.

    PubMed

    Balfanz, Sabine; Ehling, Petra; Wachten, Sebastian; Jordan, Nadine; Erber, Joachim; Mujagic, Samir; Baumann, Arnd

    2012-06-01

    The second messenger cAMP has a pivotal role in animals' physiology and behavior. Intracellular concentrations of cAMP are balanced by cAMP-synthesizing adenylyl cyclases (ACs) and cAMP-cleaving phosphodiesterases. Knowledge about ACs in the honeybee (Apis mellifera) is rather limited and only an ortholog of the vertebrate AC3 isoform has been functionally characterized, so far. Employing bioinformatics and functional expression we characterized two additional honeybee genes encoding membrane-bound (tm)ACs. The proteins were designated AmAC2t and AmAC8. Unlike the common structure of tmACs, AmAC2t lacks the first transmembrane domain. Despite this unusual topography, AmAC2t-activity could be stimulated by norepinephrine and NKH477 with EC(50s) of 0.07 μM and 3 μM. Both ligands stimulated AmAC8 with EC(50s) of 0.24 μM and 3.1 μM. In brain cryosections, intensive staining of mushroom bodies was observed with specific antibodies against AmAC8, an expression pattern highly reminiscent of the Drosophila rutabaga AC. In a current release of the honeybee genome database we identified three additional tmAC- and one soluble AC-encoding gene. These results suggest that (1) the AC-gene family in honeybees is comparably large as in other species, and (2) based on the restricted expression of AmAC8 in mushroom bodies, this enzyme might serve important functions in honeybee behavior. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. Evidence for functional pre-coupled complexes of receptor heteromers and adenylyl cyclase.

    PubMed

    Navarro, Gemma; Cordomí, Arnau; Casadó-Anguera, Verónica; Moreno, Estefanía; Cai, Ning-Sheng; Cortés, Antoni; Canela, Enric I; Dessauer, Carmen W; Casadó, Vicent; Pardo, Leonardo; Lluís, Carme; Ferré, Sergi

    2018-03-28

    G protein-coupled receptors (GPCRs), G proteins and adenylyl cyclase (AC) comprise one of the most studied transmembrane cell signaling pathways. However, it is unknown whether the ligand-dependent interactions between these signaling molecules are based on random collisions or the rearrangement of pre-coupled elements in a macromolecular complex. Furthermore, it remains controversial whether a GPCR homodimer coupled to a single heterotrimeric G protein constitutes a common functional unit. Using a peptide-based approach, we here report evidence for the existence of functional pre-coupled complexes of heteromers of adenosine A 2A receptor and dopamine D 2 receptor homodimers coupled to their cognate Gs and Gi proteins and to subtype 5 AC. We also demonstrate that this macromolecular complex provides the necessary frame for the canonical Gs-Gi interactions at the AC level, sustaining the ability of a Gi-coupled GPCR to counteract AC activation mediated by a Gs-coupled GPCR.

  11. Purification and assay of cell-invasive form of calmodulin-sensitive adenylyl cyclase from Bordetella pertussis

    SciTech Connect

    Masure, H.R.; Donovan, M.G.; Storm, D.R.

    1991-01-01

    An invasive form of the CaM-sensitive adenylyl cyclase from Bordetella pertussis can be isolated from bacterial culture supernatants. This isolation is achieved through the use of QAE-Sephadex anion-exchange chromatography. It has been demonstrated that the addition of exogenous Ca{sup 2}{sup +} to the anion-exchange gradient buffers will affect elution from the column and will thereby affect the isolation of invasive adenylyl cyclase. This is probably due to a Ca2(+)-dependent interaction of the catalytic subunit with another component in the culture supernatant. Two peaks of adenylyl cyclase activity are obtained. The Pk1 adenylyl cyclase preparation is able to cause significant increasesmore » in intracellular cAMP levels in animal cells. This increase occurs rapidly and in a dose-dependent manner in both N1E-115 mouse neuroblastoma cells and human erythrocytes. The Pk2 adenylyl cyclase has catalytic activity but is not cell invasive. This material can serve, therefore, as a control to ensure that the cAMP which is measured is, indeed, intracellular. A second control is to add exogenous CaM to the Pk1 adenylyl cyclase preparation. The 45-kDa catalytic subunit-CaM complex is not cell invasive. Although the mechanism for membrane translocation of the adenylyl cyclase is unknown, there is evidence that the adenylyl cyclase enters animal cells by a mechanism distinct from receptor-mediated endocytosis. Calmodulin-sensitive adenylyl cyclase activity can be removed from preparations of the adenylyl cyclase that have been subjected to SDS-polyacrylamide gel electrophoresis. This property of the enzyme has enabled purification of the catalytic subunit to apparent homogeneity. The purified catalytic subunit from culture supernatants has a predicted molecular weight of 45,000. This polypeptide interacts directly with Ca{sup 2}{sup +} and this interaction may be important for its invasion into animal cells.« less

  12. Mice Overexpressing Type 1 Adenylyl Cyclase Show Enhanced Spatial Memory Flexibility in the Absence of Intact Synaptic Long-Term Depression

    ERIC Educational Resources Information Center

    Zhang, Ming; Wang, Hongbing

    2013-01-01

    There is significant interest in understanding the contribution of intracellular signaling and synaptic substrates to memory flexibility, which involves new learning and suppression of obsolete memory. Here, we report that enhancement of Ca[superscript 2+]-stimulated cAMP signaling by overexpressing type 1 adenylyl cyclase (AC1) facilitated…

  13. Structural Basis for Inhibition of Mammalian Adenylyl Cyclase by Calcium

    SciTech Connect

    Mou, Tung-Chung; Masada, Nanako; Cooper, Dermot M.F.

    2009-09-11

    Type V and VI mammalian adenylyl cyclases (AC5, AC6) are inhibited by Ca{sup 2+} at both sub- and supramicromolar concentration. This inhibition may provide feedback in situations where cAMP promotes opening of Ca{sup 2+} channels, allowing fine control of cardiac contraction and rhythmicity in cardiac tissue where AC5 and AC6 predominate. Ca{sup 2+} inhibits the soluble AC core composed of the C1 domain of AC5 (VC1) and the C2 domain of AC2 (IIC2). As observed for holo-AC5, inhibition is biphasic, showing 'high-affinity' (K{sub i} = {approx}0.4 {mu}M) and 'low-affinity' (K{sub i} = {approx}100 {mu}M) modes of inhibition. At micromolar concentration,more » Ca{sup 2+} inhibition is nonexclusive with respect to pyrophosphate (PP{sub i}), a noncompetitive inhibitor with respect to ATP, but at >100 {mu}M Ca{sup 2+}, inhibition appears to be exclusive with respect to PP{sub i}. The 3.0 {angstrom} resolution structure of G{alpha}s{center_dot}GTP{gamma}S/forskolin-activated VC1:IIC2 crystals soaked in the presence of ATP{alpha}S and 8 {mu}M free Ca{sup 2+} contains a single, loosely coordinated metal ion. ATP soaked into VC1:IIC2 crystals in the presence of 1.5 mM Ca{sup 2+} is not cyclized, and two calcium ions are observed in the 2.9 {angstrom} resolution structure of the complex. In both of the latter complexes VC1:IIC2 adopts the 'open', catalytically inactive conformation characteristic of the apoenzyme, in contrast to the 'closed', active conformation seen in the presence of ATP analogues and Mg{sup 2+} or Mn{sup 2+}. Structures of the pyrophosphate (PP{sub i}) complex with 10 mM Mg{sup 2+} (2.8 {angstrom}) or 2 mM Ca{sup 2+} (2.7 {angstrom}) also adopt the open conformation, indicating that the closed to open transition occurs after cAMP release. In the latter complexes, Ca{sup 2+} and Mg{sup 2+} bind only to the high-affinity 'B' metal site associated with substrate/product stabilization. Ca{sup 2+} thus stabilizes the inactive conformation in both ATP- and PP

  14. Gi proteins regulate adenylyl cyclase activity independent of receptor activation.

    PubMed

    Melsom, Caroline Bull; Ørstavik, Øivind; Osnes, Jan-Bjørn; Skomedal, Tor; Levy, Finn Olav; Krobert, Kurt Allen

    2014-01-01

    Despite the view that only β2- as opposed to β1-adrenoceptors (βARs) couple to G(i), some data indicate that the β1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes. We used the Gs-selective (R,R)- and the Gs- and G(i)-activating (R,S)-fenoterol to selectively activate β2ARs (β1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats. PTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response. Together, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic G(i) and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents.

  15. Gi Proteins Regulate Adenylyl Cyclase Activity Independent of Receptor Activation

    PubMed Central

    Melsom, Caroline Bull; Ørstavik, Øivind; Osnes, Jan-Bjørn; Skomedal, Tor; Levy, Finn Olav; Krobert, Kurt Allen

    2014-01-01

    Background and purpose Despite the view that only β2- as opposed to β1-adrenoceptors (βARs) couple to Gi, some data indicate that the β1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes. Experimental approach We used the Gs-selective (R,R)- and the Gs- and Gi-activating (R,S)-fenoterol to selectively activate β2ARs (β1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats. Key results PTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response. Conclusions and implications Together, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic Gi and Gs activity upon AC towards Gs

  16. Expression of adenylyl cyclase types III and VI in human hyperfunctioning thyroid nodules.

    PubMed

    Celano, M; Arturi, F; Presta, I; Bruno, R; Scarpelli, D; Calvagno, M G; Cristofaro, C; Bulotta, S; Giannasio, P; Sacco, R; Filetti, S; Russo, D

    2003-05-30

    Hyperfunctioning thyroid nodules are characterized by the presence of spontaneous somatic mutations responsible for constitutive activation of the cAMP pathway. However, alterations affecting other elements of the cAMP signaling system may counteract the effects of the mutations. In this study, the expression of the adenylyl cyclase (AC) types III and VI was investigated by Western blot in 18 hyperfunctioning thyroid nodules; in 12 samples, we also assessed the presence of TSH receptor (TSHR) or gsp mutations and levels of AC VI and III mRNA. We found that the expression of nodular AC VI (but not AC III) was significantly lower (85.1% of normal, P=0.014) than the expression of both adenylyl cycles types of perinodular tissue from the same patients. Slightly, but not significant differences were detected in nodules with or without mutations and AC protein levels generally showed correlation with the levels of the transcripts detected by RT-PCR. In addition, AC III and AC VI expression levels within a given nodule were characterized by a significant positive correlation. These findings indicate that a diminished expression of AC type VI may be part of the mechanisms occurring in the hyperfunctioning nodules, independently of the presence of TSHR or gsp mutations, which influence the resulting phenotype.

  17. An adenylyl cyclase gene (NlAC9) influences growth and fecundity in the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

    USDA-ARS?s Scientific Manuscript database

    The cAMP/PKA intracellular signaling pathway is launched by adenylyl cyclase (AC) conversion of adenosine triphosphate (ATP) to 3', 5'-cyclic AMP (cAMP) and cAMP-dependent activation of PKA. Although this pathway is very well known in insect physiology, there is little to no information on it in som...

  18. The diurnal oscillation of MAP (mitogen-activated protein) kinase and adenylyl cyclase activities in the hippocampus depends on the suprachiasmatic nucleus.

    PubMed

    Phan, Trongha X; Phan, Trongha H; Chan, Guy C-K; Sindreu, Carlos B; Eckel-Mahan, Kristin L; Storm, Daniel R

    2011-07-20

    Consolidation of hippocampus-dependent memory is dependent on activation of the cAMP/Erk/MAPK (mitogen-activated protein kinase) signal transduction pathway in the hippocampus. Recently, we discovered that adenylyl cyclase and MAPK activities undergo a circadian oscillation in the hippocampus and that inhibition of this oscillation impairs contextual memory. This suggests the interesting possibility that the persistence of hippocampus-dependent memory depends upon the reactivation of MAPK in the hippocampus during the circadian cycle. A key unanswered question is whether the circadian oscillation of this signaling pathway is intrinsic to the hippocampus or is driven by the master circadian clock in the suprachiasmatic nucleus (SCN). To address this question, we ablated the SCN of mice by electrolytic lesion and examined hippocampus-dependent memory as well as adenylyl cyclase and MAPK activities. Electrolytic lesion of the SCN 2 d after training for contextual fear memory reduced contextual memory measured 2 weeks after training, indicating that maintenance of contextual memory depends on the SCN. Spatial memory was also compromised in SCN-lesioned mice. Furthermore, the diurnal oscillation of adenylyl cyclase and MAPK activities in the hippocampus was destroyed by lesioning of the SCN. These data suggest that hippocampus-dependent long-term memory is dependent on the SCN-controlled oscillation of the adenylyl cyclase/MAPK pathway in the hippocampus.

  19. Overexpression of the Type 1 Adenylyl Cyclase in the Forebrain Leads to Deficits of Behavioral Inhibition

    PubMed Central

    Cao, Hong; Saraf, Amit; Zweifel, Larry S.

    2015-01-01

    The type 1 adenylyl cyclase (AC1) is an activity-dependent, calcium-stimulated adenylyl cyclase expressed in the nervous system that is implicated in memory formation. We examined the locomotor activity, and impulsive and social behaviors of AC1+ mice, a transgenic mouse strain overexpressing AC1 in the forebrain. Here we report that AC1+ mice exhibit hyperactive behaviors and demonstrate increased impulsivity and reduced sociability. In contrast, AC1 and AC8 double knock-out mice are hypoactive, and exhibit increased sociability and reduced impulsivity. Interestingly, the hyperactivity of AC1+ mice can be corrected by valproate, a mood-stabilizing drug. These data indicate that increased expression of AC1 in the forebrain leads to deficits in behavioral inhibition. PMID:25568126

  20. Learning defects in Drosophila growth restricted chico mutants are caused by attenuated adenylyl cyclase activity.

    PubMed

    Naganos, Shintaro; Ueno, Kohei; Horiuchi, Junjiro; Saitoe, Minoru

    2016-04-06

    Reduced insulin/insulin-like growth factor signaling (IIS) is a major cause of symmetrical intrauterine growth retardation (IUGR), an impairment in cell proliferation during prenatal development that results in global growth defects and mental retardation. In Drosophila, chico encodes the only insulin receptor substrate. Similar to other animal models of IUGR, chico mutants have defects in global growth and associative learning. However, the physiological and molecular bases of learning defects caused by chico mutations, and by symmetrical IUGR, are not clear. In this study, we found that chico mutations impair memory-associated synaptic plasticity in the mushroom bodies (MBs), neural centers for olfactory learning. Mutations in chico reduce expression of the rutabaga-type adenylyl cyclase (rut), leading to decreased cAMP synthesis in the MBs. Expressing a rut (+) transgene in the MBs restores memory-associated plasticity and olfactory associative learning in chico mutants, without affecting growth. Thus chico mutations disrupt olfactory learning, at least in part, by reducing cAMP signaling in the MBs. Our results suggest that some cognitive defects associated with reduced IIS may occur, independently of developmental defects, from acute reductions in cAMP signaling.

  1. Established and potential physiological roles of bicarbonate-sensing soluble adenylyl cyclase (sAC) in aquatic animals

    PubMed Central

    Tresguerres, Martin; Barott, Katie L.; Barron, Megan E.; Roa, Jinae N.

    2014-01-01

    Soluble adenylyl cyclase (sAC) is a recently recognized source of the signaling molecule cyclic AMP (cAMP) that is genetically and biochemically distinct from the classic G-protein-regulated transmembrane adenylyl cyclases (tmACs). Mammalian sAC is distributed throughout the cytoplasm and it may be present in the nucleus and inside mitochondria. sAC activity is directly stimulated by HCO3−, and sAC has been confirmed to be a HCO3− sensor in a variety of mammalian cell types. In addition, sAC can functionally associate with carbonic anhydrases to act as a de facto sensor of pH and CO2. The two catalytic domains of sAC are related to HCO3−-regulated adenylyl cyclases from cyanobacteria, suggesting the cAMP pathway is an evolutionarily conserved mechanism for sensing CO2 levels and/or acid/base conditions. Reports of sAC in aquatic animals are still limited but are rapidly accumulating. In shark gills, sAC senses blood alkalosis and triggers compensatory H+ absorption. In the intestine of bony fishes, sAC modulates NaCl and water absorption. And in sea urchin sperm, sAC may participate in the initiation of flagellar movement and in the acrosome reaction. Bioinformatics and RT-PCR results reveal that sAC orthologs are present in most animal phyla. This review summarizes the current knowledge on the physiological roles of sAC in aquatic animals and suggests additional functions in which sAC may be involved. PMID:24574382

  2. Identification of photoactivated adenylyl cyclases in Naegleria australiensis and BLUF-containing protein in Naegleria fowleri.

    PubMed

    Yasukawa, Hiro; Sato, Aya; Kita, Ayaka; Kodaira, Ken-Ichi; Iseki, Mineo; Takahashi, Tetsuo; Shibusawa, Mami; Watanabe, Masakatsu; Yagita, Kenji

    2013-01-01

    Complete genome sequencing of Naegleria gruberi has revealed that the organism encodes polypeptides similar to photoactivated adenylyl cyclases (PACs). Screening in the N. australiensis genome showed that the organism also encodes polypeptides similar to PACs. Each of the Naegleria proteins consists of a "sensors of blue-light using FAD" domain (BLUF domain) and an adenylyl cyclase domain (AC domain). PAC activity of the Naegleria proteins was assayed by comparing sensitivities of Escherichia coli cells heterologously expressing the proteins to antibiotics in a dark condition and a blue light-irradiated condition. Antibiotics used in the assays were fosfomycin and fosmidomycin. E. coli cells expressing the Naegleria proteins showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light, indicating that the proteins functioned as PACs in the bacterial cells. Analysis of the N. fowleri genome revealed that the organism encodes a protein bearing an amino acid sequence similar to that of BLUF. A plasmid expressing a chimeric protein consisting of the BLUF-like sequence found in N. fowleri and the adenylyl cyclase domain of N. gruberi PAC was constructed to determine whether the BLUF-like sequence functioned as a sensor of blue light. E. coli cells expressing a chimeric protein showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light. These experimental results indicated that the sequence similar to the BLUF domain found in N. fowleri functioned as a sensor of blue light.

  3. The propensity for consuming ethanol in Drosophila requires rutabaga adenylyl cyclase expression within mushroom body neurons

    PubMed Central

    Xu, Shiyu; Chan, Tammy; Shah, Vruntant; Zhang, Shixing; Pletcher, Scott D.; Roman, Gregg

    2012-01-01

    Alcohol activates reward systems through an unknown mechanism, in some cases leading to alcohol abuse and dependence. Herein, we utilized a two-choice Capillary Feeding assay to address the neural and molecular basis for ethanol self-administration in Drosophila melanogaster. Wild-type Drosophila demonstrates a significant preference for food containing between 5 and 15% ethanol. Preferred ethanol self-administration does not appear to be due to caloric advantage, nor due to perceptual biases, suggesting a hedonic bias for ethanol exists in Drosophila. Interestingly, rutabaga adenylyl cyclase expression within intrinsic mushroom body neurons is necessary for robust ethanol self-administration. The expression of rutabaga in mushroom bodies is also required for both appetitive and aversive olfactory associative memories, suggesting that reinforced behavior has an important role in the ethanol self-administration in Drosophila. However, rutabaga expression is required more broadly within the mushroom bodies for the preference for ethanol-containing food than for olfactory memories reinforced by sugar reward. Together these data implicate cAMP signaling and behavioral reinforcement for preferred ethanol self-administration in Drosophila melanogaster. PMID:22624869

  4. Calcium influx through L-type channels attenuates skeletal muscle contraction via inhibition of adenylyl cyclases.

    PubMed

    Menezes-Rodrigues, Francisco Sandro; Pires-Oliveira, Marcelo; Duarte, Thiago; Paredes-Gamero, Edgar Julian; Chiavegatti, Tiago; Godinho, Rosely Oliveira

    2013-11-15

    Skeletal muscle contraction is triggered by acetylcholine induced release of Ca(2+) from sarcoplasmic reticulum. Although this signaling pathway is independent of extracellular Ca(2+), L-type voltage-gated calcium channel (Cav) blockers have inotropic effects on frog skeletal muscles which occur by an unknown mechanism. Taking into account that skeletal muscle fiber expresses Ca(+2)-sensitive adenylyl cyclase (AC) isoforms and that cAMP is able to increase skeletal muscle contraction force, we investigated the role of Ca(2+) influx on mouse skeletal muscle contraction and the putative crosstalk between extracellular Ca(2+) and intracellular cAMP signaling pathways. The effects of Cav blockers (verapamil and nifedipine) and extracellular Ca(2+) chelator EGTA were evaluated on isometric contractility of mouse diaphragm muscle under direct electrical stimulus (supramaximal voltage, 2 ms, 0.1 Hz). Production of cAMP was evaluated by radiometric assay while Ca(2+) transients were assessed by confocal microscopy using L6 cells loaded with fluo-4/AM. Ca(2+) channel blockers verapamil and nifedipine had positive inotropic effect, which was mimicked by removal of extracellular Ca(+2) with EGTA or Ca(2+)-free Tyrode. While phosphodiesterase inhibitor IBMX potentiates verapamil positive inotropic effect, it was abolished by AC inhibitors SQ22536 and NYK80. Finally, the inotropic effect of verapamil was associated with increased intracellular cAMP content and mobilization of intracellular Ca(2+), indicating that positive inotropic effects of Ca(2+) blockers depend on cAMP formation. Together, our results show that extracellular Ca(2+) modulates skeletal muscle contraction, through inhibition of Ca(2+)-sensitive AC. The cross-talk between extracellular calcium and cAMP-dependent signaling pathways appears to regulate the extent of skeletal muscle contraction responses. © 2013 Published by Elsevier B.V.

  5. Adenylyl cyclase type 9 gene polymorphisms are associated with asthma and allergy in Brazilian children.

    PubMed

    Teixeira, Helena M P; Alcantara-Neves, Neuza M; Barreto, Maurício; Figueiredo, Camila A; Costa, Ryan S

    2017-02-01

    Asthma is a chronic inflammatory disease of the respiratory tract. This heterogeneous disease is caused by the interaction of interindividual genetic variability and environmental factors. The gene adenylyl cyclase type 9 (ADCY9) encodes a protein called adenylyl cyclase (AC), responsible for producing the second messenger cyclic AMP (cAMP). cAMP is produced by T regulatory cells and is involved in the down-regulation of T effector cells. Failures in cAMP production may be related to an imbalance in the regulatory immune response, leading to immune-mediated diseases, such as allergic disorders. The aim of this study was to investigate how polymorphisms in the ADCY9 are associated with asthma and allergic markers. The study comprised 1309 subjects from the SCAALA (Social Changes Asthma and Allergy in Latin America) program. Genotyping was accomplished using the Illumina 2.5 Human Omni bead chip. Logistic regression was used to assess the association between allergy markers and ADCY9 variation in PLINK 1.07 software with adjustments for sex, age, helminth infection and ancestry markers. The ADCY9 candidate gene was associated with different phenotypes, such as asthma, specific IgE, skin prick test, and cytokine production. Among 133 markers analyzed, 29 SNPs where associated with asthma and allergic markers in silico analysis revealed the functional impact of the 6 SNPs on ADCY9 expression. It can be concluded that polymorphisms in the ADCY9 gene are significantly associated with asthma and/or allergy markers. We believe that such polymorphisms may lead to increased expression of adenylyl cyclase with a consequent increase in immunoregulatory activity. Therefore, these SNPs may offer an impact on the occurrence of these conditions in admixture population from countries such as Brazil. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Distinct Mechanisms of Calmodulin Binding and Regulation of Adenylyl Cyclases 1 and 8

    PubMed Central

    2012-01-01

    Calmodulin (CaM), by mediating the stimulation of the activity of two adenylyl cyclases (ACs), plays a key role in integrating the cAMP and Ca2+ signaling systems. These ACs, AC1 and AC8, by decoding discrete Ca2+ signals can contribute to fine-tuning intracellular cAMP dynamics, particularly in neurons where they predominate. CaM comprises an α-helical linker separating two globular regions at the N-terminus and the C-terminus that each bind two Ca2+ ions. These two lobes have differing affinities for Ca2+, and they can interact with target proteins independently. This study explores previous indications that the two lobes of CaM can regulate AC1 and AC8 differently and thereby yield different responses to cellular transitions in [Ca2+]i. We first compared by glutathione S-transferase pull-down assays and offline nanoelectrospray ionization mass spectrometry the interaction of CaM and Ca2+-binding deficient mutants of CaM with the internal CaM binding domain (CaMBD) of AC1 and the two terminal CaMBDs of AC8. We then examined the influence of these three CaMBDs on Ca2+ binding by native and mutated CaM in stopped-flow experiments to quantify their interactions. The three CaMBDs show quite distinct interactions with the two lobes of CaM. These findings establish the critical kinetic differences between the mechanisms of Ca2+-CaM activation of AC1 and AC8, which may underpin their different physiological roles. PMID:22971080

  7. AmTAR2: Functional characterization of a honeybee tyramine receptor stimulating adenylyl cyclase activity.

    PubMed

    Reim, Tina; Balfanz, Sabine; Baumann, Arnd; Blenau, Wolfgang; Thamm, Markus; Scheiner, Ricarda

    2017-01-01

    The biogenic monoamines norepinephrine and epinephrine regulate important physiological functions in vertebrates. Insects such as honeybees do not synthesize these neuroactive substances. Instead, they employ octopamine and tyramine for comparable physiological functions. These biogenic amines activate specific guanine nucleotide-binding (G) protein-coupled receptors (GPCRs). Based on pharmacological data obtained on heterologously expressed receptors, α- and β-adrenergic-like octopamine receptors are better activated by octopamine than by tyramine. Conversely, GPCRs forming the type 1 tyramine receptor clade (synonymous to octopamine/tyramine receptors) are better activated by tyramine than by octopamine. More recently, receptors were characterized which are almost exclusively activated by tyramine, thus forming an independent type 2 tyramine receptor clade. Functionally, type 1 tyramine receptors inhibit adenylyl cyclase activity, leading to a decrease in intracellular cAMP concentration ([cAMP] i ). Type 2 tyramine receptors can mediate Ca 2+ signals or both Ca 2+ signals and effects on [cAMP] i . We here provide evidence that the honeybee tyramine receptor 2 (AmTAR2), when heterologously expressed in flpTM cells, exclusively causes an increase in [cAMP] i . The receptor displays a pronounced preference for tyramine over octopamine. Its activity can be blocked by a series of established antagonists, of which mianserin and yohimbine are most efficient. The functional characterization of two tyramine receptors from the honeybee, AmTAR1 (previously named AmTYR1) and AmTAR2, which respond to tyramine by changing cAMP levels in opposite direction, is an important step towards understanding the actions of tyramine in honeybee behavior and physiology, particularly in comparison to the effects of octopamine. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Forskolin photoaffinity labels with specificity for adenylyl cyclase and the glucose transporter

    SciTech Connect

    Morris, D.I.; Robbins, J.D.; Ruoho, A.E.

    1991-07-15

    Two photolabels, N-(3-(4-azido-3-125I-phenyl)-propionamide)-6- aminoethylcarbamylforskolin(125I-6-AIPP-Fsk) and N-(3-(4-azido-3-125I-phenyl)propionamide)-7-aminoethylcarbamyl-7- desacetylforskolin (125I-7-AIPP-Fsk) were synthesized with specific activities of 2200 Ci/mmol and used to label adenylyl cyclase and the glucose transporter. The affinities of the photolabels for adenylyl cyclase were determined by their inhibition of (3H)forskolin binding to bovine brain membranes. 6-AIPP-Fsk and 7-AIPP-Fsk inhibited (3H)forskolin binding with IC50 values of 15 nM and 200 nM, respectively. 125I-6-AIPP-Fsk labeled a 115-kDa protein in control and GTP {gamma} S-preactivated bovine brain membranes. This labeling was inhibited by forskolin but not by 1,9-dideoxyforskolin or cytochalasin B. 125I-6-AIPP-Fsk labeling of partially purified adenylyl cyclase was inhibited by forskolinmore » but not by 1,9-dideoxyforskolin. 125I-7-AIPP-Fsk specifically labeled a 45-kDa protein and not a 115-kDa protein in control and GTP {gamma} S-preactivated brain membranes. This labeling was inhibited by forskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose but not cytochalasin E or L-glucose. Human erythrocyte membranes were photolyzed with 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk. 125I-7-AIPP-Fsk, but not 125I-6-AIPP-Fsk, strongly labeled a broad 45-70-kDa band. Forskolin, 7-bromoacetyl-7-desacetylforskolin, 1,9-dideoxyforskolin, cytochalasin B, and D-glucose, but not cytochalasin E or L-glucose, inhibited 125I-7-AIPP-Fsk labeling of the 45-70-kDa band. 125I-6-AIPP-Fsk and 125I-7-AIPP-Fsk are high affinity photolabels with specificity for adenylyl cyclase and the glucose transporter, respectively.« less

  9. Bicarbonate disruption of the pulmonary endothelial barrier via activation of endogenous soluble adenylyl cyclase, isoform 10

    PubMed Central

    Obiako, Boniface; Calchary, Wendy; Xu, Ningyong; Kunstadt, Ryan; Richardson, Bianca; Nix, Jessica

    2013-01-01

    It is becoming increasingly apparent that cAMP signals within the pulmonary endothelium are highly compartmentalized, and this compartmentalization is critical to maintaining endothelial barrier integrity. Studies demonstrate that the exogenous soluble bacterial toxin, ExoY, and heterologous expression of the forskolin-stimulated soluble mammalian adenylyl cyclase (AC) chimera, sACI/II, elevate cytosolic cAMP and disrupt the pulmonary microvascular endothelial barrier. The barrier-disruptive effects of cytosolic cAMP generated by exogenous soluble ACs are in contrast to the barrier-protective effects of subplasma membrane cAMP generated by transmembrane AC, which strengthens endothelial barrier integrity. Endogenous soluble AC isoform 10 (AC10 or commonly known as sAC) lacks transmembrane domains and localizes within the cytosolic compartment. AC10 is uniquely activated by bicarbonate to generate cytosolic cAMP, yet its role in regulation of endothelial barrier integrity has not been addressed. Here we demonstrate that, within the pulmonary circulation, AC10 is expressed in pulmonary microvascular endothelial cells (PMVECs) and pulmonary artery endothelial cells (PAECs), yet expression in PAECs is lower. Furthermore, pulmonary endothelial cells selectively express bicarbonate cotransporters. While extracellular bicarbonate generates a phosphodiesterase 4-sensitive cAMP pool in PMVECs, no such cAMP response is detected in PAECs. Finally, addition of extracellular bicarbonate decreases resistance across the PMVEC monolayer and increases the filtration coefficient in the isolated perfused lung above osmolality controls. Collectively, these findings suggest that PMVECs have a bicarbonate-sensitive cytosolic cAMP pool that disrupts endothelial barrier integrity. These studies could provide an alternative mechanism for the controversial effects of bicarbonate correction of acidosis of acute respiratory distress syndrome patients. PMID:23686854

  10. Impact of divalent metal ions on regulation of adenylyl cyclase isoforms by forskolin analogs.

    PubMed

    Erdorf, Miriam; Mou, Tung-Chung; Seifert, Roland

    2011-12-01

    Mammalian membranous adenylyl cyclases (mACs) play an important role in transmembrane signalling events in almost every cell and represent an interesting drug target. Forskolin (FS) is an invaluable research tool, activating AC isoforms 1-8. However, there is a paucity of AC isoform-selective FS analogs. Therefore, we examined the effects of FS and six FS derivatives on recombinant ACs 1, 2 and 5, representing members of different mAC families. Correlations of the pharmacological properties of the different AC isoforms revealed pronounced differences between ACs 1, 2 and 5. Additionally, potencies and efficacies of FS derivatives changed for any given AC isoform, depending on the metal ion, Mg(2+) or Mn(2+). The most striking effects of Mg(2+) and Mn(2+) on the diterpene profile were observed for AC2 where the large inhibitory effect of BODIPY-FS in the presence of Mg(2+) was considerably reduced in the presence of Mn(2+). Sequence alignment and docking experiments confirmed an exceptional position of AC2 compared to ACs 1 and 5 with respect to the structural environment of the catalytic core and cation-dependent diterpene effects. In conclusion, mAC isoforms 1, 2 and 5 exhibit a distinct pharmacological diterpene profile, depending on the divalent cation present. mAC crystal structures and modelling/docking studies provided an explanation for the pharmacological differences between the AC isoforms. Our study constitutes an important step towards the development of isoform-specific diterpenes exhibiting stimulatory or inhibitory effects. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Opioid and GABAB receptors differentially couple to an adenylyl cyclase/protein kinase A downstream effector after chronic morphine treatment

    PubMed Central

    Bagley, Elena E.

    2014-01-01

    Opioids are intensely addictive, and cessation of their chronic use is associated with a highly aversive withdrawal syndrome. A cellular hallmark of withdrawal is an opioid sensitive protein kinase A-dependent increase in GABA transporter-1 (GAT-1) currents in periaqueductal gray (PAG) neurons. Elevated GAT-1 activity directly increases GABAergic neuronal excitability and synaptic GABA release, which will enhance GABAergic inhibition of PAG output neurons. This reduced activity of PAG output neurons to several brain regions, including the hypothalamus and medulla, contributes to many of the PAG-mediated signs of opioid withdrawal. The GABAB receptor agonist baclofen reduces some of the PAG mediated signs of opioid withdrawal. Like the opioid receptors the GABAB receptor is a Gi/Go coupled G-protein coupled receptor. This suggests it could be modulating GAT-1 activity in PAG neurons through its inhibition of the adenylyl cyclase/protein kinase A pathway. Opioid modulation of the GAT-1 activity can be detected by changes in the reversal potential of opioid membrane currents. We found that when opioids are reducing the GAT-1 cation conductance and increasing the GIRK conductance the opioid agonist reversal potential is much more negative than Ek. Using this approach for GABAB receptors we show that the GABAB receptor agonist, baclofen, does not couple to inhibition of GAT-1 currents during opioid withdrawal. It is possible this differential signaling of the two Gi/Go coupled G-protein coupled receptors is due to the strong compartmentalization of the GABAB receptor that does not favor signaling to the adenylyl cyclase/protein kinase A/GAT-1 pathway. This highlights the importance of studying the effects of G-protein coupled receptors in native tissue with endogenous G-protein coupled receptors and the full complement of relevant proteins and signaling molecules. This study suggests that baclofen reduces opioid withdrawal symptoms through a non-GAT-1 effector. PMID

  12. Opioid and GABAB receptors differentially couple to an adenylyl cyclase/protein kinase A downstream effector after chronic morphine treatment.

    PubMed

    Bagley, Elena E

    2014-01-01

    Opioids are intensely addictive, and cessation of their chronic use is associated with a highly aversive withdrawal syndrome. A cellular hallmark of withdrawal is an opioid sensitive protein kinase A-dependent increase in GABA transporter-1 (GAT-1) currents in periaqueductal gray (PAG) neurons. Elevated GAT-1 activity directly increases GABAergic neuronal excitability and synaptic GABA release, which will enhance GABAergic inhibition of PAG output neurons. This reduced activity of PAG output neurons to several brain regions, including the hypothalamus and medulla, contributes to many of the PAG-mediated signs of opioid withdrawal. The GABAB receptor agonist baclofen reduces some of the PAG mediated signs of opioid withdrawal. Like the opioid receptors the GABAB receptor is a Gi/Go coupled G-protein coupled receptor. This suggests it could be modulating GAT-1 activity in PAG neurons through its inhibition of the adenylyl cyclase/protein kinase A pathway. Opioid modulation of the GAT-1 activity can be detected by changes in the reversal potential of opioid membrane currents. We found that when opioids are reducing the GAT-1 cation conductance and increasing the GIRK conductance the opioid agonist reversal potential is much more negative than E k . Using this approach for GABAB receptors we show that the GABAB receptor agonist, baclofen, does not couple to inhibition of GAT-1 currents during opioid withdrawal. It is possible this differential signaling of the two Gi/Go coupled G-protein coupled receptors is due to the strong compartmentalization of the GABAB receptor that does not favor signaling to the adenylyl cyclase/protein kinase A/GAT-1 pathway. This highlights the importance of studying the effects of G-protein coupled receptors in native tissue with endogenous G-protein coupled receptors and the full complement of relevant proteins and signaling molecules. This study suggests that baclofen reduces opioid withdrawal symptoms through a non-GAT-1 effector.

  13. Soluble adenylyl cyclase is an acid-base sensor in epithelial base-secreting cells.

    PubMed

    Roa, Jinae N; Tresguerres, Martin

    2016-08-01

    Blood acid-base regulation by specialized epithelia, such as gills and kidney, requires the ability to sense blood acid-base status. Here, we developed primary cultures of ray (Urolophus halleri) gill cells to study mechanisms for acid-base sensing without the interference of whole animal hormonal regulation. Ray gills have abundant base-secreting cells, identified by their noticeable expression of vacuolar-type H(+)-ATPase (VHA), and also express the evolutionarily conserved acid-base sensor soluble adenylyl cyclase (sAC). Exposure of cultured cells to extracellular alkalosis (pH 8.0, 40 mM HCO3 (-)) triggered VHA translocation to the cell membrane, similar to previous reports in live animals experiencing blood alkalosis. VHA translocation was dependent on sAC, as it was blocked by the sAC-specific inhibitor KH7. Ray gill base-secreting cells also express transmembrane adenylyl cyclases (tmACs); however, tmAC inhibition by 2',5'-dideoxyadenosine did not prevent alkalosis-dependent VHA translocation, and tmAC activation by forskolin reduced the abundance of VHA at the cell membrane. This study demonstrates that sAC is a necessary and sufficient sensor of extracellular alkalosis in ray gill base-secreting cells. In addition, this study indicates that different sources of cAMP differentially modulate cell biology. Copyright © 2016 the American Physiological Society.

  14. Nicotine-induced activation of soluble adenylyl cyclase participates in ion transport regulation in mouse tracheal epithelium.

    PubMed

    Hollenhorst, Monika I; Lips, Katrin S; Kummer, Wolfgang; Fronius, Martin

    2012-11-27

    Functional nicotinic acetylcholine receptors (nAChR) have been identified in airway epithelia and their location in the apical and basolateral membrane makes them targets for acetylcholine released from neuronal and non-neuronal sources. One function of nAChR in airway epithelia is their involvement in the regulation of transepithelial ion transport by activation of chloride and potassium channels. However, the mechanisms underlying this nicotine-induced activation of ion transport are not fully elucidated. Thus, the aim of this study was to investigate the involvement of adenylyl cyclases in the nicotine-induced ion current in mouse tracheal epithelium. To evaluate the nicotine-mediated changes of transepithelial ion transport processes electrophysiological Ussing chamber measurements were applied and nicotine-induced ion currents were recorded in the absence and presence of adenylyl cyclase inhibitors. The ion current changes induced by nicotine (100 μM, apical) were not altered in the presence of high doses of atropine (25 μM, apical and basolateral), underlining the involvement of nAChR. Experiments with the transmembrane adenylyl cyclase inhibitor 2'5'-dideoxyadenosine (50 μM, apical and basolateral) and the soluble adenylyl cyclase inhibitor KH7 (10 μM, apical and basolateral) both reduced the nicotine-mediated ion current to a similar extent. Yet, a statistically significant reduction was obtained only in the experiments with KH7. This study indicates that nicotine binding to nAChR in mouse tracheal epithelium activates transepithelial ion transport involving adenylyl cyclase activity. This might be important for novel therapeutic strategies targeting epithelial ion transport mediated by the non-neuronal cholinergic system. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. pH sensing via bicarbonate-regulated “soluble” adenylyl cyclase (sAC)

    PubMed Central

    Rahman, Nawreen; Buck, Jochen; Levin, Lonny R.

    2013-01-01

    Soluble adenylyl cyclase (sAC) is a source of the second messenger cyclic adenosine 3′, 5′ monophosphate (cAMP). sAC is directly regulated by bicarbonate (HCO−3) ions. In living cells, HCO−3 ions are in nearly instantaneous equilibrium with carbon dioxide (CO2) and pH due to the ubiquitous presence of carbonic anhydrases. Numerous biological processes are regulated by CO2, HCO−3, and/or pH, and in a number of these, sAC has been shown to function as a physiological CO2/HCO3/pH sensor. In this review, we detail the known pH sensing functions of sAC, and we discuss two highly-studied, pH-dependent pathways in which sAC might play a role. PMID:24324443

  16. Disruption of type 3 adenylyl cyclase expression in the hypothalamus leads to obesity

    PubMed Central

    Cao, Hong; Chen, Xuanmao; Yang, Yimei; Storm, Daniel R

    2016-01-01

    Evidence from human studies and transgenic mice lacking the type 3 adenylyl cyclase (AC3) indicates that AC3 plays a role in the regulation of body weight. It is unknown in which brain region AC3 exerts such an effect. We examined the role of AC3 in the hypothalamus for body weight control using a floxed AC3 mouse strain. Here, we report that AC3 flox/flox mice became obese after the administration of AAV-CRE-GFP into the hypothalamus. Both male and female AC3 floxed mice showed heavier body weight than AAV-GFP injected control mice. Furthermore, mice with selective ablation of AC3 expression in the ventromedial hypothalamus also showed increased body weight and food consumption. Our results indicated that AC3 in the hypothalamus regulates energy balance. PMID:27942392

  17. Identification of an adenylyl cyclase inhibitor for treating neuropathic and inflammatory pain.

    PubMed

    Wang, Hansen; Xu, Hui; Wu, Long-Jun; Kim, Susan S; Chen, Tao; Koga, Kohei; Descalzi, Giannina; Gong, Bo; Vadakkan, Kunjumon I; Zhang, Xuehan; Kaang, Bong-Kiun; Zhuo, Min

    2011-01-12

    Neuropathic pain, often caused by nerve injury, is commonly observed among patients with different diseases. Because its basic mechanisms are poorly understood, effective medications are limited. Previous investigations of basic pain mechanisms and drug discovery efforts have focused mainly on early sensory neurons such as dorsal root ganglion and spinal dorsal horn neurons, and few synaptic-level studies or new drugs are designed to target the injury-related cortical plasticity that accompanies neuropathic pain. Our previous work has demonstrated that calcium-stimulated adenylyl cyclase 1 (AC1) is critical for nerve injury-induced synaptic changes in the anterior cingulate cortex. Through rational drug design and chemical screening, we have identified a lead candidate AC1 inhibitor, NB001, which is relatively selective for AC1 over other adenylate cyclase isoforms. Using a variety of behavioral tests and toxicity studies, we have found that NB001, when administered intraperitoneally or orally, has an analgesic effect in animal models of neuropathic pain, without any apparent side effects. Our study thus shows that AC1 could be a productive therapeutic target for neuropathic pain and describes a new agent for the possible treatment of neuropathic pain.

  18. H{sub 2}S induces vasoconstriction of rat cerebral arteries via cAMP/adenylyl cyclase pathway

    SciTech Connect

    Li, Sen; Ping, Na-na; Cao, Lei, E-mail: leicao@mail.xjtu.edu.cn

    2015-12-15

    Hydrogen sulfide (H{sub 2}S), traditionally known for its toxic effects, is now involved in regulating vascular tone. Here we investigated the vasoconstrictive effect of H{sub 2}S on cerebral artery and the underlying mechanism. Sodium hydrosulfide (NaHS), a donor of H{sub 2}S, concentration-dependently induced vasoconstriction on basilar artery, which was enhanced in the presence of isoprenaline, a β-adrenoceptor agonist or forskolin, an adenylyl cyclase activator. Administration of NaHS attenuated the vasorelaxant effects of isoprenaline or forskolin. Meanwhile, the NaHS-induced vasoconstriction was diminished in the presence of 8B-cAMP, an analog of cAMP, but was not affected by Bay K-8644, a selective L-typemore » Ca{sup 2+} channel agonist. These results could be explained by the revised effects of NaHS on isoprenaline-induced cAMP elevation and forskolin-stimulated adenylyl cyclase activity. Additionally, NaHS-induced vasoconstriction was enhanced by removing the endothelium or in the presence of L-NAME, an inhibitor of nitric oxide synthase. L-NAME only partially attenuated the effect of NaHS which was given together with forskolin on the pre-contracted artery. In conclusion, H{sub 2}S induces vasoconstriction of cerebral artery via, at least in part, cAMP/adenylyl cyclase pathway. - Highlights: • The vasoactivity effect of NaHS, a donor of H{sub 2}S, was studied on rat cerebral arteries. • H{sub 2}S induces a constriction, not a relaxant effect on basilar arteries. • The vasoconstrictive effect is invovled in inhibiting adenylyl cyclase to reduce cAMP levels. • The vasoconstriction is partially antagonized by NO, and does not necessarily act via NO pathway.« less

  19. Adenylyl cyclase A mRNA localized at the back of cells is actively translated in live chemotaxing Dictyostelium.

    PubMed

    Wang, Weiye; Chen, Song; Das, Satarupa; Losert, Wolfgang; Parent, Carole A

    2018-05-04

    Dictyostelium discoideum cells transport adenylyl cyclase A (ACA)-containing vesicles to the back of polarized cells to relay exogenous cAMP signals during chemotaxis. Fluorescence in situ hybridization (FISH) experiments showed that ACA mRNA is also asymmetrically distributed at the back of polarized cells. By using the MS2 bacteriophage system, we now visualize the distribution of ACA mRNA in live chemotaxing cells. We found that the ACA mRNA localization is not dependent on the translation of the protein product and requires multiple cis-acting elements within the ACA-coding sequence. We show that ACA mRNA is associated with actively translating ribosomes and is transported along microtubules towards the back of cells. By monitoring the recovery of ACA-YFP after photobleaching, we observed that local translation of ACA-YFP occurs at the back of cells. These data represent a novel functional role for localized translation in the relay of chemotactic signals during chemotaxis. © 2018. Published by The Company of Biologists Ltd.

  20. Metabolic communication between astrocytes and neurons via bicarbonate-responsive soluble adenylyl cyclase.

    PubMed

    Choi, Hyun B; Gordon, Grant R J; Zhou, Ning; Tai, Chao; Rungta, Ravi L; Martinez, Jennifer; Milner, Teresa A; Ryu, Jae K; McLarnon, James G; Tresguerres, Martin; Levin, Lonny R; Buck, Jochen; MacVicar, Brian A

    2012-09-20

    Astrocytes are proposed to participate in brain energy metabolism by supplying substrates to neurons from their glycogen stores and from glycolysis. However, the molecules involved in metabolic sensing and the molecular pathways responsible for metabolic coupling between different cell types in the brain are not fully understood. Here we show that a recently cloned bicarbonate (HCO₃⁻) sensor, soluble adenylyl cyclase (sAC), is highly expressed in astrocytes and becomes activated in response to HCO₃⁻ entry via the electrogenic NaHCO₃ cotransporter (NBC). Activated sAC increases intracellular cAMP levels, causing glycogen breakdown, enhanced glycolysis, and the release of lactate into the extracellular space, which is subsequently taken up by neurons for use as an energy substrate. This process is recruited over a broad physiological range of [K⁺](ext) and also during aglycemic episodes, helping to maintain synaptic function. These data reveal a molecular pathway in astrocytes that is responsible for brain metabolic coupling to neurons. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Metabolic Communication between Astrocytes and Neurons via Bicarbonate-Responsive Soluble Adenylyl Cyclase

    PubMed Central

    Choi, Hyun B.; Gordon, Grant R.J.; Zhou, Ning; Tai, Chao; Rungta, Ravi L.; Martinez, Jennifer; Milner, Teresa A.; Ryu, Jae K.; McLarnon, James G.; Tresguerres, Martin; Levin, Lonny R.; Buck, Jochen; MacVicar, Brian A.

    2013-01-01

    SUMMARY Astrocytes are proposed to participate in brain energy metabolism by supplying substrates to neurons from their glycogen stores and from glycolysis. However, the molecules involved in metabolic sensing and the molecular pathways responsible for metabolic coupling between different cell types in the brain are not fully understood. Here we show that a recently cloned bicarbonate (HCO3−) sensor, soluble adenylyl cyclase (sAC), is highly expressed in astrocytes and becomes activated in response to HCO3− entry via the electrogenic NaHCO3 cotransporter (NBC). Activated sAC increases intracellular cAMP levels, causing glycogen breakdown, enhanced glycolysis, and the release of lactate into the extracellular space, which is subsequently taken up by neurons for use as an energy substrate. This process is recruited over a broad physiological range of [K+]ext and also during aglycemic episodes, helping to maintain synaptic function. These data reveal a molecular pathway in astrocytes that is responsible for brain metabolic coupling to neurons. PMID:22998876

  2. Adenylyl cyclase localization to the uropod of aggregating Dictyostelium cells requires RacC

    PubMed Central

    Wang, C.; Jung, D.; Cao, Z.; Chung, C. Y.

    2015-01-01

    The localization of adenylyl cyclase A (ACA) to uropod of cells is required for the stream formation during Dictyostelium development. RacC is a Dictyostelium orthologue of Cdc42. We identified a streaming defect of racC− cells as they are clearly less polarized and form smaller and fragmented streams. ACA-YFP is mainly associated with intracellular vesicular structures, but not with the plasma membrane in racC− cells. racC− cells have a slightly higher number of vesicles than Ax3 cells, suggesting that the defect of ACA trafficking is not simply due to the lack of vesicle formation. While the ACA-YFP vesicles traveled with an average velocity of 9.1 µm/min in Ax3 cells, a slow and diffusional movement without direction with an average velocity of 4 µm/min was maintained in racC− cells. Images acquired by using total internal reflection fluorescence (TIRF) microscopy and fluorescence recovery after photobleaching (FRAP) analysis revealed that a significantly decreased number of ACA-YFP vesicles appeared near the cell membrane, indicating a defect in ACA-YFP vesicle trafficking. These results suggest an important role of RacC in the rapid and directional movements of ACA vesicles on microtubules to the plasma membrane, especially to the back of polarized cell. PMID:26315268

  3. Activity of adenylyl cyclase and protein kinase A contributes to morphine-induced spinal apoptosis.

    PubMed

    Lim, Grewo; Wang, Shuxing; Lim, Jeong-Ae; Mao, Jianren

    2005-12-02

    Our previous study has shown that chronic morphine exposure induces neuronal apoptosis within the spinal cord dorsal horn; however, the mechanisms of morphine-induced apoptosis remain unclear. Here we examined whether adenylyl cyclase (AC) and protein kinase A (PKA) would play a role in this process. Intrathecal morphine regimen (10 microg, twice daily x 7 days) that resulted in antinociceptive tolerance induced spinal apoptosis as revealed by in situ terminal deoxynucleotidyl transferase (TdT)-UTP-biotin nick end labeling (TUNEL). The TUNEL-positive cells were detected primarily in the superficial laminae of the spinal cord dorsal horn, which was associated with an increase in the expression of activated caspase-3 and mitogen-activated protein kinase (MAPK) within the same spinal region. Co-administration of morphine with a broad AC inhibitor (ddA), a PKA inhibitor (H89), or a MAPK inhibitor (PD98059) substantially reduced the number of TUNEL-positive cells, as compared with the morphine alone group. The results indicate that the spinal AC and PKA pathway through intracellular MAPK may be contributory to the cellular mechanisms of morphine-induced apoptosis.

  4. Activation of the adenylyl cyclase/cyclic AMP/protein kinase A pathway in endothelial cells exposed to cyclic strain

    NASA Technical Reports Server (NTRS)

    Cohen, C. R.; Mills, I.; Du, W.; Kamal, K.; Sumpio, B. E.

    1997-01-01

    The aim of this study was to assess the involvement of the adenylyl cyclase/cyclic AMP/protein kinase A pathway (AC) in endothelial cells (EC) exposed to different levels of mechanical strain. Bovine aortic EC were seeded to confluence on flexible membrane-bottom wells. The membranes were deformed with either 150 mm Hg (average 10% strain) or 37.5 mm Hg (average 6% strain) vacuum at 60 cycles per minute (0.5 s strain; 0.5 s relaxation) for 0-60 min. The results demonstrate that at 10% average strain (but not 6% average strain) there was a 1.5- to 2.2-fold increase in AC, cAMP, and PKA activity by 15 min when compared to unstretched controls. Further studies revealed an increase in cAMP response element binding protein in EC subjected to the 10% average strain (but not 6% average strain). These data support the hypothesis that cyclic strain activates the AC/cAMP/PKA signal transduction pathway in EC which may occur by exceeding a strain threshold and suggest that cyclic strain may stimulate the expression of genes containing cAMP-responsive promoter elements.

  5. Palmitoylation targets AKAP79 protein to lipid rafts and promotes its regulation of calcium-sensitive adenylyl cyclase type 8.

    PubMed

    Delint-Ramirez, Ilse; Willoughby, Debbie; Hammond, Gerald R V; Hammond, Gerald V R; Ayling, Laura J; Cooper, Dermot M F

    2011-09-23

    PKA anchoring proteins (AKAPs) optimize the efficiency of cAMP signaling by clustering interacting partners. Recently, AKAP79 has been reported to directly bind to adenylyl cyclase type 8 (AC8) and to regulate its responsiveness to store-operated Ca(2+) entry (SOCE). Although AKAP79 is well targeted to the plasma membrane via phospholipid associations with three N-terminal polybasic regions, recent studies suggest that AKAP79 also has the potential to be palmitoylated, which may specifically allow it to target the lipid rafts where AC8 resides and is regulated by SOCE. In this study, we have addressed the role of palmitoylation of AKAP79 using a combination of pharmacological, mutagenesis, and cell biological approaches. We reveal that AKAP79 is palmitoylated via two cysteines in its N-terminal region. This palmitoylation plays a key role in targeting the AKAP to lipid rafts in HEK-293 cells. Mutation of the two critical cysteines results in exclusion of AKAP79 from lipid rafts and alterations in its membrane diffusion behavior. This is accompanied by a loss of the ability of AKAP79 to regulate SOCE-dependent AC8 activity in intact cells and decreased PKA-dependent phosphorylation of raft proteins, including AC8. We conclude that palmitoylation plays a key role in the targeting and action of AKAP79. This novel property of AKAP79 adds an unexpected regulatory and targeting option for AKAPs, which may be exploited in the cellular context.

  6. Fluorogenic Green-Inside Red-Outside (GIRO) Labeling Approach Reveals Adenylyl Cyclase-Dependent Control of BKα Surface Expression

    PubMed Central

    2015-01-01

    The regulation of surface levels of protein is critical for proper cell function and influences properties including cell adhesion, ion channel contributions to current flux, and the sensitivity of surface receptors to ligands. Here we demonstrate a two-color labeling system in live cells using a single fluorogen activating peptide (FAP) based fusion tag, which enables the rapid and simultaneous quantification of surface and internal proteins. In the nervous system, BK channels can regulate neural excitability and neurotransmitter release, and the surface trafficking of BK channels can be modulated by signaling cascades and assembly with accessory proteins. Using this labeling approach, we examine the dynamics of BK channel surface expression in HEK293 cells. Surface pools of the pore-forming BKα subunit were stable, exhibiting a plasma membrane half-life of >10 h. Long-term activation of adenylyl cyclase by forskolin reduced BKα surface levels by 30%, an effect that could not be attributed to increased bulk endocytosis of plasma membrane proteins. This labeling approach is compatible with microscopic imaging and flow cytometry, providing a solid platform for examining protein trafficking in living cells. PMID:26301573

  7. A novel cytosolic regulator, Pianissimo, is required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase in Dictyostelium

    PubMed Central

    Chen, Mei-Yu; Long, Yu; Devreotes, Peter N.

    1997-01-01

    Genetic analysis was applied to identify novel genes involved in G protein-linked pathways controlling development. Using restriction enzyme-mediated integration (REMI), we have identified a new gene, Pianissimo (PiaA), involved in cAMP signaling in Dictyostelium discoideum. PiaA encodes a 130-kD cytosolic protein required for chemoattractant receptor and G protein-mediated activation of the 12 transmembrane domain adenylyl cyclase. In piaA− null mutants, neither chemoattractant stimulation of intact cells nor GTPγS treatment of lysates activates the enzyme; constitutive expression of PiaA reverses these defects. Cytosols of wild-type cells that contain Pia protein reconstitute the GTPγS stimulation of adenylyl cyclase activity in piaA− lysates, indicating that Pia is directly involved in the activation. Pia and CRAC, a previously identified cytosolic regulator, are both essential for activation of the enzyme as lysates of crac− piaA− double mutants require both proteins for reconstitution. Homologs of PiaA are found in Saccharomyces cerevisiae and Schizosaccaromyces pombe; disruption of the S. cerevisiae homolog results in lethality. We propose that homologs of Pia and similar modes of regulation of these ubiquitous G protein-linked pathways are likely to exist in higher eukaryotes. PMID:9389653

  8. Isolated dorsal root ganglion neurones inhibit receptor-dependent adenylyl cyclase activity in associated glial cells

    PubMed Central

    Ng, KY; Yeung, BHS; Wong, YH; Wise, H

    2013-01-01

    Background and Purpose Hyper-nociceptive PGE2 EP4 receptors and prostacyclin (IP) receptors are present in adult rat dorsal root ganglion (DRG) neurones and glial cells in culture. The present study has investigated the cell-specific expression of two other Gs-protein coupled hyper-nociceptive receptor systems: β-adrenoceptors and calcitonin gene-related peptide (CGRP) receptors in isolated DRG cells and has examined the influence of neurone–glial cell interactions in regulating adenylyl cyclase (AC) activity. Experimental Approach Agonist-stimulated AC activity was determined in mixed DRG cell cultures from adult rats and compared with activity in DRG neurone-enriched cell cultures and pure DRG glial cell cultures. Key Results Pharmacological analysis showed the presence of Gs-coupled β2-adrenoceptors and CGRP receptors, but not β1-adrenoceptors, in all three DRG cell preparations. Agonist-stimulated AC activity was weakest in DRG neurone-enriched cell cultures. DRG neurones inhibited IP receptor-stimulated glial cell AC activity by a process dependent on both cell–cell contact and neurone-derived soluble factors, but this is unlikely to involve purine or glutamine receptor activation. Conclusions and Implications Gs-coupled hyper-nociceptive receptors are readily expressed on DRG glial cells in isolated cell cultures and the activity of CGRP, EP4 and IP receptors, but not β2-adrenoceptors, in glial cells is inhibited by DRG neurones. Studies using isolated DRG cells should be aware that hyper-nociceptive ligands may stimulate receptors on glial cells in addition to neurones, and that variable numbers of neurones and glial cells will influence absolute measures of AC activity and affect downstream functional responses. PMID:22924655

  9. Regulation of anterior chamber drainage by bicarbonate-sensitive soluble adenylyl cyclase in the ciliary body.

    PubMed

    Lee, Yong S; Tresguerres, Martin; Hess, Kenneth; Marmorstein, Lihua Y; Levin, Lonny R; Buck, Jochen; Marmorstein, Alan D

    2011-12-02

    Glaucoma is a leading cause of blindness affecting as many as 2.2 million Americans. All current glaucoma treatment strategies aim to reduce intraocular pressure (IOP). IOP results from the resistance to drainage of aqueous humor (AH) produced by the ciliary body in a process requiring bicarbonate. Once secreted into the anterior chamber, AH drains from the eye via two pathways: uveoscleral and pressure-dependent or conventional outflow (C(t)). Modulation of "inflow" and "outflow" pathways is thought to occur via distinct, local mechanisms. Mice deficient in the bicarbonate channel bestrophin-2 (Best2), however, exhibit a lower IOP despite an increase in AH production. Best2 is expressed uniquely in nonpigmented ciliary epithelial (NPE) cells providing evidence for a bicarbonate-dependent communicative pathway linking inflow and outflow. Here, we show that bicarbonate-sensitive soluble adenylyl cyclase (sAC) is highly expressed in the ciliary body in NPE cells, but appears to be absent from drainage tissues. Pharmacologic inhibition of sAC in mice causes a significant increase in IOP due to a decrease in C(t) with no effect on inflow. In mice deficient in sAC IOP is elevated, and C(t) is decreased relative to wild-type mice. Pharmacologic inhibition of sAC did not alter IOP or C(t) in sAC-deficient mice. Based on these data we propose that the ciliary body can regulate C(t) and that sAC serves as a critical sensor of bicarbonate in the ciliary body regulating the secretion of substances into the AH that govern outflow facility independent of pressure.

  10. Solubilization of adenylyl cyclase from human myometrium in a alphas-coupled form.

    PubMed

    Bajo, Ana M; Prieto, Juan C; Valenzuela, Pedro; Martinez, Pilar; Guijarro, Luis G

    2003-08-01

    Adenylyl cyclase (AC) was extracted from human myometrium with either non-ionic (Lubrol-PX or Triton X-100) or zwitterionic (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, CHAPS) detergents. The soluble enzyme was stimulated by forskolin, a hydrophobic activator, in the presence of Mg2+ indicating that the catalytic subunit had not been damaged after solubilization. The enzyme was also activated by 5'-guanylyl imidodiphosphate (Gpp(NH)p) showing that the catalytic unit was not separated from stimulatory guanine nucleotide binding protein (Gs) during the extraction. Both activators showed different effects on the stimulatory efficacy and potency of AC activity solobulized with detergents. Gel filtration of Lubrol-PX and CHAPS extracts over a Sepharose CL-2B column partially resolved AC and its complexes. The chromatographic profile for Lubrol-solubilized AC presented a main peak of about 200 kDa whereas CHAPS-solubilized AC showed a dominant peak of about 1100 kDa. The heterodisperse peaks obtained revealed that the catalytic AC subunit was not separated from Gs proteins after gel filtration, and that AC could be associated with other cellular proteins. When Lubrol extract was submitted to anionic-exchange chromatography, the enzyme was purified about 7.5 fold (enzymatic activity of 48.1 pmol/min/mg of protein). The catalytic subunit was co-eluted with both AC-activating proteins Galphas large (52.2 kDa) and Galphas small (48.7 kDa). This is the first demonstration of the stable physical association of AC with both alphas subunits of G proteins in human myometrium.

  11. Soluble Adenylyl Cyclase Is Required for Retinal Ganglion Cell and Photoreceptor Differentiation

    PubMed Central

    Shaw, Peter X.; Fang, Jiahua; Sang, Alan; Wang, Yan; Kapiloff, Michael S.; Goldberg, Jeffrey L.

    2016-01-01

    Purpose We have previously demonstrated that soluble adenylyl cyclase (sAC) is necessary for retinal ganglion cell (RGC) survival and axon growth. Here, we further investigate the role of sAC in neuronal differentiation during retinal development. Methods Chx10 or Math5 promoter-driven Cre-Lox recombination were used to conditionally delete sAC from early and intermediate retinal progenitor cells during retinal development. We examined cell type–specific markers expressed by retinal cells to estimate their relative numbers and characterize retinal laminar morphology by immunofluorescence in adult and newborn mice. Results Retinal ganglion cell and amacrine cell markers were significantly lower in the retinas of adult Math5cre/sACfl/fl and Chx10cre/sACfl/fl mice than in those of wild-type controls. The effect on RGC development was detectable as early as postnatal day 1 and deleting sAC in either Math5- or Chx10-expressing retinal progenitor cells also reduced nerve fiber layer thickness into adulthood. The thickness of the photoreceptor layer was slightly but statistically significantly decreased in both the newborn Chx10cre/sACfl/fl and Math5cre/sACfl/fl mice, but this reduction and abnormal morphology persisted in the adults in only the Chx10cre/sACfl/fl mice. Conclusions sAC plays an important role in the early retinal development of RGCs as well as in the development of amacrine cells and to a lesser degree photoreceptors. PMID:27679853

  12. A mitochondrial CO2-adenylyl cyclase-cAMP signalosome controls yeast normoxic cytochrome c oxidase activity

    PubMed Central

    Hess, Kenneth C.; Liu, Jingjing; Manfredi, Giovanni; Mühlschlegel, Fritz A.; Buck, Jochen; Levin, Lonny R.; Barrientos, Antoni

    2014-01-01

    Mitochondria, the major source of cellular energy in the form of ATP, respond to changes in substrate availability and bioenergetic demands by employing rapid, short-term, metabolic adaptation mechanisms, such as phosphorylation-dependent protein regulation. In mammalian cells, an intramitochondrial CO2-adenylyl cyclase (AC)-cyclic AMP (cAMP)-protein kinase A (PKA) pathway regulates aerobic energy production. One target of this pathway involves phosphorylation of cytochrome c oxidase (COX) subunit 4-isoform 1 (COX4i1), which modulates COX allosteric regulation by ATP. However, the role of the CO2-sAC-cAMP-PKA signalosome in regulating COX activity and mitochondrial metabolism and its evolutionary conservation remain to be fully established. We show that in Saccharomyces cerevisiae, normoxic COX activity measured in the presence of ATP is 55% lower than in the presence of ADP. Moreover, the adenylyl cyclase Cyr1 activity is present in mitochondria, and it contributes to the ATP-mediated regulation of COX through the normoxic subunit Cox5a, homologue of human COX4i1, in a bicarbonate-sensitive manner. Furthermore, we have identified 2 phosphorylation targets in Cox5a (T65 and S43) that modulate its allosteric regulation by ATP. These residues are not conserved in the Cox5b-containing hypoxic enzyme, which is not regulated by ATP. We conclude that across evolution, a CO2-sAC-cAMP-PKA axis regulates normoxic COX activity.—Hess, K. C., Liu, J., Manfredi, G., Mühlschlegel, F. A., Buck, J., Levin, L. R., Barrientos, A. A mitochondrial CO2-adenylyl cyclase-cAMP signalosome controls yeast normoxic cytochrome c oxidase activity. PMID:25002117

  13. Three alpha-subunits of heterotrimeric G proteins and an adenylyl cyclase have distinct roles in fruiting body development in the homothallic fungus Sordaria macrospora.

    PubMed

    Kamerewerd, Jens; Jansson, Malin; Nowrousian, Minou; Pöggeler, Stefanie; Kück, Ulrich

    2008-09-01

    Sordaria macrospora, a self-fertile filamentous ascomycete, carries genes encoding three different alpha-subunits of heterotrimeric G proteins (gsa, G protein Sordaria alpha subunit). We generated knockout strains for all three gsa genes (Deltagsa1, Deltagsa2, and Deltagsa3) as well as all combinations of double mutants. Phenotypic analysis of single and double mutants showed that the genes for Galpha-subunits have distinct roles in the sexual life cycle. While single mutants show some reduction of fertility, double mutants Deltagsa1Deltagsa2 and Deltagsa1Deltagsa3 are completely sterile. To test whether the pheromone receptors PRE1 and PRE2 mediate signaling via distinct Galpha-subunits, two recently generated Deltapre strains were crossed with all Deltagsa strains. Analyses of the corresponding double mutants revealed that compared to GSA2, GSA1 is a more predominant regulator of a signal transduction cascade downstream of the pheromone receptors and that GSA3 is involved in another signaling pathway that also contributes to fruiting body development and fertility. We further isolated the gene encoding adenylyl cyclase (AC) (sac1) for construction of a knockout strain. Analyses of the three DeltagsaDeltasac1 double mutants and one Deltagsa2Deltagsa3Deltasac1 triple mutant indicate that SAC1 acts downstream of GSA3, parallel to a GSA1-GSA2-mediated signaling pathway. In addition, the function of STE12 and PRO41, two presumptive signaling components, was investigated in diverse double mutants lacking those developmental genes in combination with the gsa genes. This analysis was further completed by expression studies of the ste12 and pro41 transcripts in wild-type and mutant strains. From the sum of all our data, we propose a model for how different Galpha-subunits interact with pheromone receptors, adenylyl cyclase, and STE12 and thus cooperatively regulate sexual development in S. macrospora.

  14. Bacterial effector binds host cell adenylyl cyclase to potentiate Gαs-dependent cAMP production

    PubMed Central

    Pulliainen, Arto T.; Pieles, Kathrin; Brand, Cameron S.; Hauert, Barbara; Böhm, Alex; Quebatte, Maxime; Wepf, Alexander; Gstaiger, Matthias; Aebersold, Ruedi; Dessauer, Carmen W.; Dehio, Christoph

    2012-01-01

    Subversion of host organism cAMP signaling is an efficient and widespread mechanism of microbial pathogenesis. Bartonella effector protein A (BepA) of vasculotumorigenic Bartonella henselae protects the infected human endothelial cells against apoptotic stimuli by elevation of cellular cAMP levels by an as yet unknown mechanism. Here, adenylyl cyclase (AC) and the α-subunit of the AC-stimulating G protein (Gαs) were identified as potential cellular target proteins for BepA by gel-free proteomics. Results of the proteomics screen were evaluated for physical and functional interaction by: (i) a heterologous in vivo coexpression system, where human AC activity was reconstituted under the regulation of Gαs and BepA in Escherichia coli; (ii) in vitro AC assays with membrane-anchored full-length human AC and recombinant BepA and Gαs; (iii) surface plasmon resonance experiments; and (iv) an in vivo fluorescence bimolecular complementation-analysis. The data demonstrate that BepA directly binds host cell AC to potentiate the Gαs-dependent cAMP production. As opposed to the known microbial mechanisms, such as ADP ribosylation of G protein α-subunits by cholera and pertussis toxins, the fundamentally different BepA-mediated elevation of host cell cAMP concentration appears subtle and is dependent on the stimulus of a G protein-coupled receptor-released Gαs. We propose that this mechanism contributes to the persistence of Bartonella henselae in the chronically infected vascular endothelium. PMID:22635269

  15. Pituitary adenylyl cyclase-activating peptide: A pivotal modulator of glutamatergic regulation of the suprachiasmatic circadian clock

    PubMed Central

    Chen, Dong; Buchanan, Gordon F.; Ding, Jian M.; Hannibal, Jens; Gillette, Martha U.

    1999-01-01

    The circadian clock in the suprachiasmatic nucleus (SCN) of the hypothalamus organizes behavioral rhythms, such as the sleep–wake cycle, on a near 24-h time base and synchronizes them to environmental day and night. Light information is transmitted to the SCN by direct retinal projections via the retinohypothalamic tract (RHT). Both glutamate (Glu) and pituitary adenylyl cyclase-activating peptide (PACAP) are localized within the RHT. Whereas Glu is an established mediator of light entrainment, the role of PACAP is unknown. To understand the functional significance of this colocalization, we assessed the effects of nocturnal Glu and PACAP on phasing of the circadian rhythm of neuronal firing in slices of rat SCN. When coadministered, PACAP blocked the phase advance normally induced by Glu during late night. Surprisingly, blocking PACAP neurotransmission, with either PACAP6–38, a specific PACAP receptor antagonist, or anti-PACAP antibodies, augmented the Glu-induced phase advance. Blocking PACAP in vivo also potentiated the light-induced phase advance of the rhythm of hamster wheel-running activity. Conversely, PACAP enhanced the Glu-induced delay in the early night, whereas PACAP6–38 inhibited it. These results reveal that PACAP is a significant component of the Glu-mediated light-entrainment pathway. When Glu activates the system, PACAP receptor-mediated processes can provide gain control that generates graded phase shifts. The relative strengths of the Glu and PACAP signals together may encode the amplitude of adaptive circadian behavioral responses to the natural range of intensities of nocturnal light. PMID:10557344

  16. CO2/HCO3−- and Calcium-regulated Soluble Adenylyl Cyclase as a Physiological ATP Sensor*

    PubMed Central

    Zippin, Jonathan H.; Chen, Yanqiu; Straub, Susanne G.; Hess, Kenneth C.; Diaz, Ana; Lee, Dana; Tso, Patrick; Holz, George G.; Sharp, Geoffrey W. G.; Levin, Lonny R.; Buck, Jochen

    2013-01-01

    The second messenger molecule cAMP is integral for many physiological processes. In mammalian cells, cAMP can be generated from hormone- and G protein-regulated transmembrane adenylyl cyclases or via the widely expressed and structurally and biochemically distinct enzyme soluble adenylyl cyclase (sAC). sAC activity is uniquely stimulated by bicarbonate ions, and in cells, sAC functions as a physiological carbon dioxide, bicarbonate, and pH sensor. sAC activity is also stimulated by calcium, and its affinity for its substrate ATP suggests that it may be sensitive to physiologically relevant fluctuations in intracellular ATP. We demonstrate here that sAC can function as a cellular ATP sensor. In cells, sAC-generated cAMP reflects alterations in intracellular ATP that do not affect transmembrane AC-generated cAMP. In β cells of the pancreas, glucose metabolism generates ATP, which corresponds to an increase in cAMP, and we show here that sAC is responsible for an ATP-dependent cAMP increase. Glucose metabolism also elicits insulin secretion, and we further show that sAC is necessary for normal glucose-stimulated insulin secretion in vitro and in vivo. PMID:24100033

  17. Transmembrane adenylyl cyclase regulates amphibian sperm motility through Protein Kinase A activation

    PubMed Central

    O’Brien, Emma D.; Krapf, Darío; Cabada, Marcelo O.; Visconti, Pablo E.; Arranz, Silvia E.

    2014-01-01

    Sperm motility is essential for achieving fertilization. In animals with external fertilization as amphibians, spermatozoa are stored in a quiescent state in the testis. Spermiation to hypotonic fertilization media triggers activation of sperm motility. Bufo arenarum sperm are immotile in artificial seminal plasma (ASP) but acquire in situ flagellar beating upon dilution. In addition to the effect of low osmolarity on sperm motility activation, we report that diffusible factors of the egg jelly coat (EW) regulate motility patterns, switching from in situ to progressive movement. The signal transduction pathway involved in amphibian sperm motility activation is mostly unknown. In the present study, we show a correlation between motility activation triggered by low osmotic pressure and activation of protein kinase A (PKA). Moreover, this is the first study to present strong evidences that point toward a role of a transmembrane adenyl-cyclase (tmAC) in the regulation of amphibian sperm motility through PKA activation. PMID:21126515

  18. Distinct pools of cAMP centre on different isoforms of adenylyl cyclase in pituitary-derived GH3B6 cells.

    PubMed

    Wachten, Sebastian; Masada, Nanako; Ayling, Laura-Jo; Ciruela, Antonio; Nikolaev, Viacheslav O; Lohse, Martin J; Cooper, Dermot M F

    2010-01-01

    Microdomains have been proposed to explain specificity in the myriad of possible cellular targets of cAMP. Local differences in cAMP levels can be generated by phosphodiesterases, which control the diffusion of cAMP. Here, we address the possibility that adenylyl cyclases, the source of cAMP, can be primary architects of such microdomains. Distinctly regulated adenylyl cyclases often contribute to total cAMP levels in endogenous cellular settings, making it virtually impossible to determine the contribution of a specific isoform. To investigate cAMP dynamics with high precision at the single-isoform level, we developed a targeted version of Epac2-camps, a cAMP sensor, in which the sensor was tagged to a catalytically inactive version of the Ca(2+)-stimulable adenylyl cyclase 8 (AC8). This sensor, and less stringently targeted versions of Epac2-camps, revealed opposite regulation of cAMP synthesis in response to Ca(2+) in GH(3)B(6) pituitary cells. Ca(2+) release triggered by thyrotropin-releasing hormone stimulated the minor endogenous AC8 species. cAMP levels were decreased by inhibition of AC5 and AC6, and simultaneous activation of phosphodiesterases, in different compartments of the same cell. These findings demonstrate the existence of distinct adenylyl-cyclase-centered cAMP microdomains in live cells and open the door to their molecular micro-dissection.

  19. Intracoronary Gene Transfer of Adenylyl Cyclase 6 in Patients With Heart Failure

    PubMed Central

    Hammond, H. Kirk; Penny, William F.; Traverse, Jay H.; Henry, Timothy D.; Watkins, Matthew W.; Yancy, Clyde W.; Sweis, Ranya N.; Adler, Eric D.; Patel, Amit N.; Murray, David R.; Ross, Robert S.; Bhargava, Valmik; Maisel, Alan; Barnard, Denise D.; Lai, N. Chin; Dalton, Nancy D.; Lee, Martin L.; Narayan, Sanjiv M.; Blanchard, Daniel G.; Gao, Mei Hua

    2017-01-01

    Importance Gene transfer has rarely been tested in randomized clinical trials. Objective To evaluate the safety and efficacy of intracoronary delivery of adenovirus 5 encoding adenylyl cyclase 6 (Ad5.hAC6) in heart failure. Design, Setting, and Participants A randomized, double-blind, placebo-controlled, phase 2 clinical trial was conducted in US medical centers (randomization occurred from July 19, 2010, to October 30, 2014). Participants 18 to 80 years with symptomatic heart failure (ischemic and nonischemic) and an ejection fraction (EF) of 40% or less were screened; 86 individuals were enrolled, and 56 were randomized. Data analysis was of the intention-to-treat population. Participants underwent exercise testing and measurement of left ventricular EF (echocardiography) and then cardiac catheterization, where left ventricular pressure development (+dP/dt)and decline (−dP/dt) were recorded. Participants were randomized (3:1 ratio) to receive 1 of 5 doses of intracoronary Ad5.hAC6 or placebo. Participants underwent a second catheterization 4 weeks later for measurement of dP/dt. Exercise testing and EF were assessed 4 and 12 weeks after randomization. Interventions Intracoronary administration of Ad5.hAC6 (3.2 × 109 to 1012 virus particles) or placebo. Main Outcomes and Measures Primary end points included exercise duration and EF before and 4 and 12 weeks after randomization and peak rates of +dP/dt and −dP/dt before and 4 weeks after randomization. Fourteen placebo participants were compared (intention to treat) with 24 Ad5.hAC6 participants receiving the highest 2 doses (D4 + 5). Results Fifty-six individuals were randomized and monitored for up to 1 year. Forty-two participants (75%) received Ad5.hAC6 (mean [SE] age, 63 [1] years; EF, 30% [1%]), and 14 individuals (25%) received placebo (age, 62 [1] years; EF, 30% [2%]). Exercise duration showed no significant group differences (4 weeks, P = .27; 12 weeks, P = .47, respectively). The D4 + 5 participants

  20. Stimulation of Electro-Olfactogram Responses in the Main Olfactory Epithelia by Airflow Depend on the Type 3 Adenylyl Cyclase

    PubMed Central

    Chen, Xuanmao; Xia, Zhengui; Storm, Daniel R.

    2012-01-01

    Cilia of olfactory sensory neurons (OSN) are the primary sensory organelles for olfaction. The detection of odorants by the main olfactory epithelium (MOE) depends on coupling of odorant receptors to the type 3 adenylyl cyclase (AC3) in olfactory cilia. We monitored the effect of airflow on electro-olfactogram (EOG) responses and found that the MOE of mice can sense mechanical forces generated by airflow. The airflow-sensitive EOG response in the MOE was attenuated when cAMP was increased by odorants or by forskolin suggesting a common mechanism for airflow and odorant detection. In addition, the sensitivity to airflow was significantly impaired in the MOE from AC3−/− mice. We conclude that AC3 in the MOE is required for detecting the mechanical force of airflow, which in turn may regulate odorant perception during sniffing. PMID:23136416

  1. Three α-Subunits of Heterotrimeric G Proteins and an Adenylyl Cyclase Have Distinct Roles in Fruiting Body Development in the Homothallic Fungus Sordaria macrospora

    PubMed Central

    Kamerewerd, Jens; Jansson, Malin; Nowrousian, Minou; Pöggeler, Stefanie; Kück, Ulrich

    2008-01-01

    Sordaria macrospora, a self-fertile filamentous ascomycete, carries genes encoding three different α-subunits of heterotrimeric G proteins (gsa, G protein Sordaria alpha subunit). We generated knockout strains for all three gsa genes (Δgsa1, Δgsa2, and Δgsa3) as well as all combinations of double mutants. Phenotypic analysis of single and double mutants showed that the genes for Gα-subunits have distinct roles in the sexual life cycle. While single mutants show some reduction of fertility, double mutants Δgsa1Δgsa2 and Δgsa1Δgsa3 are completely sterile. To test whether the pheromone receptors PRE1 and PRE2 mediate signaling via distinct Gα-subunits, two recently generated Δpre strains were crossed with all Δgsa strains. Analyses of the corresponding double mutants revealed that compared to GSA2, GSA1 is a more predominant regulator of a signal transduction cascade downstream of the pheromone receptors and that GSA3 is involved in another signaling pathway that also contributes to fruiting body development and fertility. We further isolated the gene encoding adenylyl cyclase (AC) (sac1) for construction of a knockout strain. Analyses of the three ΔgsaΔsac1 double mutants and one Δgsa2Δgsa3Δsac1 triple mutant indicate that SAC1 acts downstream of GSA3, parallel to a GSA1–GSA2-mediated signaling pathway. In addition, the function of STE12 and PRO41, two presumptive signaling components, was investigated in diverse double mutants lacking those developmental genes in combination with the gsa genes. This analysis was further completed by expression studies of the ste12 and pro41 transcripts in wild-type and mutant strains. From the sum of all our data, we propose a model for how different Gα-subunits interact with pheromone receptors, adenylyl cyclase, and STE12 and thus cooperatively regulate sexual development in S. macrospora. PMID:18723884

  2. Adenylyl Cyclase 9 Polymorphisms Reveal Potential Link to HDL Function and Cardiovascular Events in Multiple Pathologies: Potential Implications in Sickle Cell Disease.

    PubMed

    Niesor, Eric J; Benghozi, Renée; Amouyel, Philippe; Ferdinand, Keith C; Schwartz, Gregory G

    2015-12-01

    Adenylyl cyclase 9 (ADCY9) mediates β2-adrenoceptor (β2-AR) signalling. Both proteins are associated with caveolae, specialized cholesterol-rich membrane substructures. Apolipoprotein A1 (ApoA1), the major protein component of high-density lipoprotein (HDL), removes cholesterol from cell membrane and caveolae and may thereby influence β2-AR signalling, shown in vitro to be modulated by cholesterol. Patients with Sickle Cell Disease (SCD) typically have low HDL and ApoA1 levels. In patients, mainly of African origin, with SCD, β2-AR activation may trigger adhesion of red blood cells to endothelial cells, leading to vascular occlusive events. Moreover, ADCY9 polymorphism is associated with risk of stroke in SCD. In recent clinical trials, ADCY9 polymorphism was found to be a discriminant factor associated with the risk of cardiovascular (CV) events in Caucasian patients treated with the HDL-raising compound dalcetrapib. We hypothesize that these seemingly disparate observations share a common mechanism related to interaction of HDL/ApoA1 and ADCY9 on β2-AR signalling. This review also raises the importance of characterizing polymorphisms that determine the response to HDL-raising and -mimicking agents in the non-Caucasian population at high risk of CV diseases and suffering from SCD. This may facilitate personalized CV treatments.

  3. Lysophosphatidic acid and adenylyl cyclase inhibitor increase proliferation of senescent human diploid fibroblasts by inhibiting adenosine monophosphate-activated protein kinase.

    PubMed

    Rhim, Ji-Heon; Jang, Ik-Soon; Song, Kye-Yong; Ha, Moon-Kyung; Cho, Sung-Chun; Yeo, Eui-Ju; Park, Sang Chul

    2008-08-01

    This study was designed to elucidate the molecular mechanism underlying lysophosphatidic acid (LPA) and adenylyl cyclase inhibitor SQ22536 (ACI)-induced senescent human diploid fibroblast (HDF) proliferation. Because adenosine monophosphate (AMP)-activated protein kinase (AMPK) is known to inhibit cell proliferation, we examined the phosphorylation status of AMPK and p53 and the expression level of p21(waf1/cip1) after treating HDFs with LPA and ACI. Phosphorylation of AMPKalpha on threonine-172 (p-Thr172-AMPKalpha) increases its catalytic activity but phosphorylation on serine-485/491 (p-Ser485/491-AMPKalpha) reduces the accessibility of the Thr172 phosphorylation site thereby inhibiting its catalytic activity. LPA increased p-Ser485/491-AMPKalpha, presumably by activating cAMP-dependent protein kinase (PKA). However, ACI reduced p-Thr172-AMPKalpha by inhibiting the LKB signaling. Our data demonstrated that both LPA and ACI inhibit the catalytic activity of AMPKalpha and p53 by differentially regulating phosphorylation of AMPKalpha, causing increased senescent cell proliferation. These findings suggest that the proliferation potential of senescent HDFs can be modulated through the regulation of the AMPK signaling pathway.

  4. Delivery of Large Heterologous Polypeptides across the Cytoplasmic Membrane of Antigen-Presenting Cells by the Bordetella RTX Hemolysin Moiety Lacking the Adenylyl Cyclase Domain

    PubMed Central

    Holubova, Jana; Jelinek, Jiri; Tomala, Jakub; Masin, Jiri; Kosova, Martina; Stanek, Ondrej; Bumba, Ladislav; Michalek, Jaroslav; Kovar, Marek; Sebo, Peter

    2012-01-01

    The Bordetella adenylate cyclase toxin-hemolysin (CyaA; also called ACT or AC-Hly) targets CD11b-expressing phagocytes and translocates into their cytosol an adenylyl cyclase (AC) that hijacks cellular signaling by conversion of ATP to cyclic AMP (cAMP). Intriguingly, insertion of large passenger peptides removes the enzymatic activity but not the cell-invasive capacity of the AC domain. This has repeatedly been exploited for delivery of heterologous antigens into the cytosolic pathway of CD11b-expressing dendritic cells by CyaA/AC− toxoids, thus enabling their processing and presentation on major histocompatibility complex (MHC) class I molecules to cytotoxic CD8+ T lymphocytes (CTLs). We produced a set of toxoids with overlapping deletions within the first 371 residues of CyaA and showed that the structure of the AC enzyme does not contain any sequences indispensable for its translocation across target cell membrane. Moreover, replacement of the AC domain (residues 1 to 371) with heterologous polypeptides of 40, 146, or 203 residues yielded CyaAΔAC constructs that delivered passenger CTL epitopes into antigen-presenting cells (APCs) and induced strong antigen-specific CD8+ CTL responses in vivo in mice and ex vivo in human peripheral blood mononuclear cell cultures. This shows that the RTX (repeats in toxin) hemolysin moiety, consisting of residues 374 to 1706 of CyaA, harbors all structural information involved in translocation of the N-terminal AC domain across target cell membranes. These results decipher the extraordinary capacity of the AC domain of CyaA to transport large heterologous cargo polypeptides into the cytosol of CD11b+ target cells and pave the way for the construction of CyaAΔAC-based polyvalent immunotherapeutic T cell vaccines. PMID:22215742

  5. RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation

    PubMed Central

    Lim, Chinten James; Spiegelman, George B.; Weeks, Gerald

    2001-01-01

    Disruption of Dictyostelium rasC, encoding a Ras subfamily protein, generated cells incapable of aggregation. While rasC expression is enriched in a cell type-specific manner during post-aggregative development, the defect in rasC– cells is restricted to aggregation and fully corrected by application of exogenous cAMP pulses. cAMP is not produced in rasC– cells stimulated by 2′-deoxy-cAMP, but is produced in response to GTPγS in cell lysates, indicating that G-protein-coupled cAMP receptor activation of adenylyl cyclase is regulated by RasC. However, cAMP-induced ERK2 phosphorylation is unaffected in rasC– cells, indicating that RasC is not an upstream activator of the mitogen-activated protein kinase required for cAMP relay. rasC– cells also exhibit reduced chemotaxis to cAMP during early development and delayed response to periodic cAMP stimuli produced by wild-type cells in chimeric mixtures. Furthermore, cAMP-induced Akt/PKB phosphorylation through a phosphatidylinositide 3-kinase (PI3K)-dependent pathway is dramatically reduced in rasC– cells, suggesting that G-protein-coupled serpentine receptor activation of PI3K is regulated by RasC. Cells lacking the RasGEF, AleA, exhibit similar defects as rasC– cells, suggesting that AleA may activate RasC. PMID:11500376

  6. RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation.

    PubMed

    Lim, C J; Spiegelman, G B; Weeks, G

    2001-08-15

    Disruption of Dictyostelium rasC, encoding a Ras subfamily protein, generated cells incapable of aggregation. While rasC expression is enriched in a cell type-specific manner during post-aggregative development, the defect in rasC(-) cells is restricted to aggregation and fully corrected by application of exogenous cAMP pulses. cAMP is not produced in rasC(-) cells stimulated by 2'-deoxy-cAMP, but is produced in response to GTPgammaS in cell lysates, indicating that G-protein-coupled cAMP receptor activation of adenylyl cyclase is regulated by RasC. However, cAMP-induced ERK2 phosphorylation is unaffected in rasC(-) cells, indicating that RasC is not an upstream activator of the mitogen-activated protein kinase required for cAMP relay. rasC(-) cells also exhibit reduced chemotaxis to cAMP during early development and delayed response to periodic cAMP stimuli produced by wild-type cells in chimeric mixtures. Furthermore, cAMP-induced Akt/PKB phosphorylation through a phosphatidylinositide 3-kinase (PI3K)-dependent pathway is dramatically reduced in rasC(-) cells, suggesting that G-protein-coupled serpentine receptor activation of PI3K is regulated by RasC. Cells lacking the RasGEF, AleA, exhibit similar defects as rasC(-) cells, suggesting that AleA may activate RasC.

  7. Adenylyl cyclase-associated protein-1/CAP1 as a biological target substrate of gelatinase B/MMP-9

    SciTech Connect

    Cauwe, Benedicte; Martens, Erik; Van den Steen, Philippe E.

    2008-09-10

    Matrix metalloproteinases (MMPs) are classically associated with the turnover of secreted structural and functional proteins. Although MMPs have been shown to process also a kaleidoscope of membrane-associated substrates, little is known about the processing of intracellular proteins by MMPs. Physiological and pathological cell apoptosis, necrosis and tumor lysis by chemotherapy, radiotherapy or immunological cytotoxicity, are examples of conditions in which an overload of intracellular proteins becomes accessible to the action of MMPs. We used a model system of dying human myelomonocytic cells to study the processing of intracellular protein substrates by gelatinase B/MMP-9 in vitro. Adenylyl cyclase-associated protein-1 or CAP1more » was identified as a novel and most efficient substrate of gelatinase B/MMP-9. The presence of CAP1 in the extracellular milieu in vivo was documented by analysis of urine of patients with systemic autoimmune diseases. Whereas no active MMP-9 could be detected in urines of healthy controls, all urine samples of patients with clinical parameters of renal failure contained activated MMP-9 and/or MMP-2. In addition, in some of these patients indications of CAP1 cleavage are observed, implying CAP1 degradation in vivo. The high turnover rate of CAP1 by MMP-9, comparable to that of gelatin as the natural extracellular substrate of this enzyme, may be critical to prevent pathological conditions associated with considerable cytolysis.« less

  8. Bicarbonate-regulated adenylyl cyclase (sAC) is a sensor that regulates pH-dependent V-ATPase recycling.

    PubMed

    Pastor-Soler, Nuria; Beaulieu, Valerie; Litvin, Tatiana N; Da Silva, Nicolas; Chen, Yanqiu; Brown, Dennis; Buck, Jochen; Levin, Lonny R; Breton, Sylvie

    2003-12-05

    Modulation of environmental pH is critical for the function of many biological systems. However, the molecular identity of the pH sensor and its interaction with downstream effector proteins remain poorly understood. Using the male reproductive tract as a model system in which luminal acidification is critical for sperm maturation and storage, we now report a novel pathway for pH regulation linking the bicarbonate activated soluble adenylyl cyclase (sAC) to the vacuolar H+ATPase (V-ATPase). Clear cells of the epididymis and vas deferens contain abundant V-ATPase in their apical pole and are responsible for acidifying the lumen. Proton secretion is regulated via active recycling of V-ATPase. Here we demonstrate that this recycling is regulated by luminal pH and bicarbonate. sAC is highly expressed in clear cells, and apical membrane accumulation of V-ATPase is triggered by a sAC-dependent rise in cAMP in response to alkaline luminal pH. As sAC is expressed in other acid/base transporting epithelia, including kidney and choroid plexus, this cAMP-dependent signal transduction pathway may be a widespread mechanism that allows cells to sense and modulate extracellular pH.

  9. An adenylyl cyclase like-9 gene (NlAC9) influences growth and fecundity in the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae).

    PubMed

    Ge, LinQuan; Gu, HaoTian; Huang, Bo; Song, Qisheng; Stanley, David; Liu, Fang; Yang, Guo-Qing; Wu, Jin-Cai

    2017-01-01

    The cAMP/PKA intracellular signaling pathway is launched by adenylyl cyclase (AC) conversion of adenosine triphosphate (ATP) to 3', 5'-cyclic AMP (cAMP) and cAMP-dependent activation of PKA. Although this pathway is very well known in insect physiology, there is little to no information on it in some very small pest insects, such as the brown planthopper (BPH), Nilaparvata lugens Stål. BPH is a destructive pest responsible for tremendous crop losses in rice cropping systems. We are investigating the potentials of novel pest management technologies from RNA interference perspective. Based on analysis of transcriptomic data, the BPH AC like-9 gene (NlAC9) was up-regulated in post-mating females, which led us to pose the hypothesis that NlAC9 is a target gene that would lead to reduced BPH fitness and populations. Targeting NlAC9 led to substantially decreased soluble ovarian protein content, yeast-like symbiont abundance, and vitellogenin gene expression, accompanied with stunted ovarian development and body size. Eggs laid were decreased and oviposition period shortened. Taken together, our findings indicated that NlAC9 exerted pronounced effects on female fecundity, growth and longevity, which strongly supports our hypothesis.

  10. Soluble adenylyl cyclase mediates mitochondrial pathway of apoptosis and ATP metabolism in oyster Crassostrea gigas exposed to elevated CO2.

    PubMed

    Wang, Xiudan; Wang, Mengqiang; Xu, Jiachao; Jia, Zhihao; Liu, Zhaoqun; Wang, Lingling; Song, Linsheng

    2017-07-01

    Ocean acidification (OA) has deleterious impacts on immune response and energy homeostasis status of Mollusca. In the present study, the apoptosis ratio of hemocytes and the adenosine triphosphate (ATP) allocation in gill tissues were determined after Pacific oysters Crassostrea gigas were exposed to elevated CO 2 environment (pH = 7.50) for 16 days.The apoptosis ratio in CO 2 exposure group (35.2%) was significantly higher (p < 0.05) than that in the control group, and the increased apoptosis ratio induced by elevated CO 2 could be significantly inhibited (p < 0.05) by KH7, a specific inhibitor of a bicarbonate sensor soluble adenylyl cyclase (sAC). After CO 2 exposure, sAC in oyster (CgsAC) was found to be clustered with mitochondria in the cytoplasm, and the pro-caspase-3 was cleaved into two small fragments. Moreover, the activities of caspase-3 and caspase-9 also increased post CO 2 exposure and these increases could be inhibited by KH7. However, the activities of caspase-8 did not change significantly compared with that in the control group. After CO 2 exposure, the ATP content in the gill increased significantly (p < 0.05) and such increase could also be inhibited by KH7. The ATP content in purified gill mitochondria decreased significantly (p < 0.05) after CO 2 exposure, which was also inhibited by KH7. These results implied that the elevated CO 2 could activate the mitochondria-CgsAC pathway of apoptosis and ATP metabolism in oyster, and this pathway played essential roles in maintaining the homeostasis and the balance of energy metabolism in response to OA. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. The structure of the regulatory domain of the adenylyl cyclase Rv1264 from Mycobacterium tuberculosis with bound oleic acid.

    PubMed

    Findeisen, Felix; Linder, Jürgen U; Schultz, Anita; Schultz, Joachim E; Brügger, Britta; Wieland, Felix; Sinning, Irmgard; Tews, Ivo

    2007-06-22

    The universal secondary messenger cAMP is produced by adenylyl cyclases (ACs). Most bacterial and all eukaryotic ACs belong to class III of six divergent classes. A class III characteristic is formation of the catalytic pocket at a dimer interface and the presence of additional regulatory domains. Mycobacterium tuberculosis possesses 15 class III ACs, including Rv1264, which is activated at acidic pH due to pH-dependent structural transitions of the Rv1264 dimer. It has been shown by X-ray crystallography that the N-terminal regulatory and C-terminal catalytic domains of Rv1264 interact in completely different ways in the active and inhibited states. Here, we report an in-depth structural and functional analysis of the regulatory domain of Rv1264. The 1.6 A resolution crystal structure shows the protein in a tight, disk-shaped dimer, formed around a helical bundle, and involving a protein chain crossover. To understand pH regulation, we determined structures at acidic and basic pH values and employed structure-based mutagenesis in the holoenzyme to elucidate regulation using an AC activity assay. It has been shown that regulatory and catalytic domains must be linked in a single protein chain. The new studies demonstrate that the length of the linker segment is decisive for regulation. Several amino acids on the surface of the regulatory domain, when exchanged, altered the pH-dependence of AC activity. However, these residues are not conserved amongst a number of related ACs. The closely related mycobacterial Rv2212, but not Rv1264, is strongly activated by the addition of fatty acids. The structure resolved the presence of a deeply embedded fatty acid, characterised as oleic acid by mass spectrometry, which may serve as a hinge. From these data, we conclude that the regulatory domain is a structural scaffold used for distinct regulatory purposes.

  12. Adenylyl cyclase 6 enhances NKCC2 expression and mediates vasopressin-induced phosphorylation of NKCC2 and NCC.

    PubMed

    Rieg, Timo; Tang, Tong; Uchida, Shinichi; Hammond, H Kirk; Fenton, Robert A; Vallon, Volker

    2013-01-01

    Arginine vasopressin (AVP) affects kidney function via vasopressin V2 receptors that are linked to activation of adenylyl cyclase (AC) and an increase in cyclic adenosine monophosphate formation. AVP/cyclic adenosine monophosphate enhance the phosphorylation of the Na-K-2Cl cotransporter (NKCC2) at serine residue 126 (pS126 NKCC2) and of the Na-Cl cotransporter (NCC) at threonine 58 (pT58 NCC). The isoform(s) of AC involved in these responses, however, were unknown. Phosphorylation of S126 NKCC2 and T58 NCC, induced by the V2 receptor agonist (1-desamino-8-D-arginine vasopressin) in wild-type mice, is lacking in knockout mice for AC isoform 6 (AC6). With regard to NKCC2 phosphorylation, the stimulatory effect of 1-desamino-8-D-AVP and the defect in AC6(-/-) mice seem to be restricted to the medullary portion of the thick ascending limb. AC6 is also a stimulator of total renal NKCC2 protein abundance in medullary and cortical thick ascending limb. Consequently, mice lacking AC6 have lower NKCC2 expression and a mild Bartter syndrome-like phenotype, including lower plasma concentrations of K+ and H+ and compensatory upregulation of NCC. Increased AC6-independent phosphorylation of NKCC2 at S126 might help to stabilize NKCC2 activity in the absence of AC6. Renal AC6 determines total NKCC2 expression and mediates vasopressin-induced NKCC2/NCC phosphorylation. These regulatory mechanisms, which are defective in AC knockout mice, are likely responsible for the observed mild Bartter syndrome. Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  13. Absorption and emission spectroscopic characterization of photo-dynamics of photoactivated adenylyl cyclase mutant bPAC-Y7F of Beggiatoa sp.

    PubMed

    Penzkofer, Alfons; Stierl, Manuela; Mathes, Tilo; Hegemann, Peter

    2014-11-01

    The photoactivated cyclase bPAC of the microbial mats bacterium Beggiatoa sp. consists of a BLUF domain and an adenylyl cyclase domain. It has strong activity of photo-induced cyclic adenylyl monophosphate (cAMP) formation and is therefore an important optogenetic tool in neuroscience applications. The SUMO-bPAC-Y7F mutant where Tyr-7 is replaced by Phe-7 in the BLUF domain has lost the typical BLUF domain photo-cycle dynamics. Instead, the investigated SUMO-bPAC-Y7F mutant consisted of three protein conformations with different triplet based photo-dynamics: (i) reversible flavin quinone (Fl) cofactor reduction to flavin semiquinone (FlH), (ii) reversible violet/near ultraviolet absorbing flavin photoproduct (FlA) formation, and (iii) irreversible red absorbing flavin photoproduct (FlC) formation. Absorption and emission spectroscopic measurements on SUMO-bPAC-Y7F were carried out before, during and after light exposure. Flavin photo-dynamics schemes are developed for the SUMO-bPAC-Y7F fractions performing photo-induced FlH, FlA, and FlC formation. Quantitative parameters of the flavin cofactor excitation, relaxation and recovery dynamics in SUMO-bPAC-Y7F are determined. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Impaired activation of adenylyl cyclase in lung of the Basenji-greyhound model of airway hyperresponsiveness: decreased numbers of high affinity beta-adrenoceptors.

    PubMed Central

    Emala, C. W.; Aryana, A.; Hirshman, C. A.

    1996-01-01

    1. To evaluate mechanisms involved in the impaired beta-adrenoceptor stimulation of adenylyl cyclase in tissues from the Basenji-greyhound (BG) dog model of airway hyperresponsiveness, we compared agonist and antagonist binding affinity of beta-adrenoceptors, beta-adrenoceptor subtypes, percentage of beta-adrenoceptors sequestered, and coupling of the beta-adrenoceptor to Gs alpha in lung membranes from BG and control mongrel dogs. We found that lung membranes from the BG dog had higher total numbers of beta-adrenoceptors with a greater percentage of receptors of the beta 2 subtype as compared to mongrel lung membranes. 2. Agonist and antagonist binding affinity and the percentage of beta-adrenoceptors sequestered were not different in BG and mongrel dog lung membranes. However, the percentage of beta-adrenoceptors in the high affinity state for agonist was decreased in BG lung membranes suggesting an uncoupling of the receptor from Gs alpha. 3. Impaired coupling between the beta-adrenoceptor and G protein documented by the decreased numbers of beta-adrenoceptors in the high affinity state in BG lung membranes, is a plausible explanation for the reduced stimulation of adenylyl cyclase and the resultant reduction in airway smooth muscle relaxation in this model. PMID:8864536

  15. Structure, signaling mechanism and regulation of the natriuretic peptide receptor guanylate cyclase.

    SciTech Connect

    Misono, K. S.; Philo, J. S.; Arakawa, T.

    2011-06-01

    Atrial natriuretic peptide (ANP) and the homologous B-type natriuretic peptide are cardiac hormones that dilate blood vessels and stimulate natriuresis and diuresis, thereby lowering blood pressure and blood volume. ANP and B-type natriuretic peptide counterbalance the actions of the renin-angiotensin-aldosterone and neurohormonal systems, and play a central role in cardiovascular regulation. These activities are mediated by natriuretic peptide receptor-A (NPRA), a single transmembrane segment, guanylyl cyclase (GC)-linked receptor that occurs as a homodimer. Here, we present an overview of the structure, possible chloride-mediated regulation and signaling mechanism of NPRA and other receptor GCs. Earlier, we determined the crystal structures ofmore » the NPRA extracellular domain with and without bound ANP. Their structural comparison has revealed a novel ANP-induced rotation mechanism occurring in the juxtamembrane region that apparently triggers transmembrane signal transduction. More recently, the crystal structures of the dimerized catalytic domain of green algae GC Cyg12 and that of cyanobacterium GC Cya2 have been reported. These structures closely resemble that of the adenylyl cyclase catalytic domain, consisting of a C1 and C2 subdomain heterodimer. Adenylyl cyclase is activated by binding of G{sub s}{alpha} to C2 and the ensuing 7{sup o} rotation of C1 around an axis parallel to the central cleft, thereby inducing the heterodimer to adopt a catalytically active conformation. We speculate that, in NPRA, the ANP-induced rotation of the juxtamembrane domains, transmitted across the transmembrane helices, may induce a similar rotation in each of the dimerized GC catalytic domains, leading to the stimulation of the GC catalytic activity.« less

  16. Adenylyl Cyclase 1 Is Required for Ethanol-Induced Locomotor Sensitization and Associated Increases in NMDA Receptor Phosphorylation and Function in the Dorsal Medial Striatum

    PubMed Central

    Bosse, Kelly E.; Oginsky, Max F.; Susick, Laura L.; Ramalingam, Sailesh; Ferrario, Carrie R.

    2017-01-01

    Neuroadaptive responses to chronic ethanol, such as behavioral sensitization, are associated with N-methyl-D-aspartate receptor (NMDAR) recruitment. Ethanol enhances GluN2B-containing NMDAR function and phosphorylation (Tyr-1472) of the GluN2B-NMDAR subunit in the dorsal medial striatum (DMS) through a protein kinase A (PKA)–dependent pathway. Ethanol-induced phosphorylation of PKA substrates is partially mediated by calcium-stimulated adenylyl cyclase 1 (AC1), which is enriched in the dorsal striatum. As such, AC1 is poised as an upstream modulator of ethanol-induced DMS neuroadaptations that promote drug responding, and thus represents a therapeutic target. Our hypothesis is that loss of AC1 activity will prevent ethanol-induced locomotor sensitization and associated DMS GluN2B-NMDAR adaptations. We evaluated AC1’s contribution to ethanol-evoked locomotor responses and DMS GluN2B-NMDAR phosphorylation and function using AC1 knockout (AC1KO) mice. Results were mechanistically validated with the AC1 inhibitor, NB001. Acute ethanol (2.0 g/kg) locomotor responses in AC1KO and wild-type (WT) mice pretreated with NB001 (10 mg/kg) were comparable to WT ethanol controls. However, repeated ethanol treatment (10 days, 2.5 g/kg) failed to produce sensitization in AC1KO or NB001 pretreated mice, as observed in WT ethanol controls, following challenge exposure (2.0 g/kg). Repeated exposure to ethanol in the sensitization procedure significantly increased pTyr-1472 GluN2B levels and GluN2B-containing NMDAR transmission in the DMS of WT mice. Loss of AC1 signaling impaired ethanol-induced increases in DMS pGluN2B levels and NMDAR-mediated transmission. Together, these data support a critical and specific role for AC1 in striatal signaling that mediates ethanol-induced behavioral sensitization, and identify GluN2B-containing NMDARs as an important AC1 target. PMID:28838956

  17. An Improved Targeted cAMP Sensor to Study the Regulation of Adenylyl Cyclase 8 by Ca2+ Entry through Voltage-Gated Channels

    PubMed Central

    Everett, Katy L.; Cooper, Dermot M. F.

    2013-01-01

    Here we describe an improved sensor with reduced pH sensitivity tethered to adenylyl cyclase (AC) 8. The sensor was used to study cAMP dynamics in the AC8 microdomain of MIN6 cells, a pancreatic β-cell line. In these cells, AC8 was activated by Ca2+ entry through L-type voltage-gated channels following depolarisation. This activation could be reconstituted in HEK293 cells co-expressing AC8 and either the α1C or α1D subunit of L-type voltage-gated Ca2+ channels. The development of this improved sensor opens the door to the study of cAMP microdomains in excitable cells that have previously been challenging due to the sensitivity of fluorescent proteins to pH changes. PMID:24086669

  18. Adenylyl cyclase-5 in the dorsal striatum function as a molecular switch for the generation of behavioral preferences for cue-directed food choices.

    PubMed

    Kim, Hannah; Kim, Tae-Kyung; Kim, Ji-Eun; Park, Jin-Young; Lee, Yunjin; Kang, Minkyung; Kim, Kyoung-Shim; Han, Pyung-Lim

    2014-11-07

    Behavioral choices in habits and innate behaviors occur automatically in the absence of conscious selection. These behaviors are not easily modified by learning. Similar types of behaviors also occur in various mental illnesses including drug addiction, obsessive-compulsive disorder, schizophrenia, and autism. However, underlying mechanisms are not clearly understood. In the present study, we investigated the molecular mechanisms regulating unconditioned preferred behaviors in food-choices. Mice lacking adenylyl cyclase-5 (AC5 KO mice), which is preferentially expressed in the dorsal striatum, consumed food pellets nearly one after another in cages. AC5 KO mice showed aversive behaviors to bitter tasting quinine, but they compulsively chose quinine-containing AC5 KO-pellets over fresh pellets. The unusual food-choice behaviors in AC5 KO mice were due to the gain of behavioral preferences for food pellets containing an olfactory cue, which wild-type mice normally ignored. Such food-choice behaviors in AC5 KO mice disappeared when whiskers were trimmed. Conversely, whisker trimming in wildtype mice induced behavioral preferences for AC5 KO food pellets, indicating that preferred food-choices were not learned through prior experience. Both AC5 KO mice and wildtype mice with trimmed whiskers had increased glutamatergic input from the barrel cortex into the dorsal striatum, resulting in an increase in the mGluR1-dependent signaling cascade. The siRNA-mediated inhibition of mGluR1 in the dorsal striatum in AC5 KO mice and wildtype mice with trimmed whiskers abolished preferred choices for AC5 KO food pellets, whereas siRNA-mediated inhibition of mGluR3 glutamate receptors in the dorsal striatum in wildtype mice induced behavioral preferences for AC5 KO food pellets, thus mimicking AC5 KO phenotypes. Our results show that the gain and loss of behavioral preferences for a specific cue-directed option were regulated by specific cellular factors in the dorsal striatum, such

  19. Partial agonist clonidine mediates alpha(2)-AR subtypes specific regulation of cAMP accumulation in adenylyl cyclase II transfected DDT1-MF2 cells.

    PubMed

    Limon-Boulez, I; Bouet-Alard, R; Gettys, T W; Lanier, S M; Maltier, J P; Legrand, C

    2001-02-01

    alpha2-Adrenergic receptor (alpha(2)-AR) activation in the pregnant rat myometrium at midterm potentiates beta(2)-AR stimulation of adenylyl cyclase (AC) via Gbetagamma regulation of the type II isoform of adenylyl cyclase. However, at term, alpha(2)-AR activation inhibits beta(2)-AR stimulation of AC. This phenomenon is associated with changes in alpha(2)-AR subtype expression (midterm alpha(2A/D)-AR > alpha(2B)-AR; term alpha(2B) >or =alpha(2A/D)-AR), without any change in ACII mRNA, suggesting that alpha(2A/D)- and alpha(2B)-AR differentially regulate beta(2)-cAMP production. To address this issue, we have stably expressed the same density of alpha(2A/D)- or alpha(2B)-AR with AC II in DDT1-MF2 cells. Clonidine (partial agonist) increased beta(2)-AR-stimulated cAMP production in alpha(2A/D)-AR-ACII transfectants but inhibited it in alpha(2B)-AR-ACII transfectants. In contrast, epinephrine (full agonist) enhanced beta(2)-stimulated ACII in both alpha(2A)- and alpha(2B)-ACII clonal cell lines. 4-Azidoanilido-[alpha-(32)P]GTP-labeling of activated G proteins indicated that, in alpha(2B)-AR transfectants, clonidine activated only Gi(2), whereas epinephrine, the full agonist, effectively coupled to Gi(2) and Gi(3). Thus, partial and full agonists selectively activate G proteins that lead to drug specific effects on effectors. Moreover, these data indicate that Gi(3) activation is required for potentiation of beta(2)-AR stimulation of AC by alpha(2A/D) and alpha(2B)-AR in DDT1-MF2 cells. This may reflect an issue of the amount of Gbetagamma released upon receptor activation and/or betagamma composition of Gi(3) versus Gi(2).

  20. Adenylyl cyclase-associated protein 1 in metastasis of squamous cell carcinoma of the head and neck and non-small cell lung cancer

    NASA Astrophysics Data System (ADS)

    Kakurina, G. V.; Kolegova, E. S.; Cheremisina, O. V.; Zavyalov, A. A.; Shishkin, D. A.; Kondakova, I. V.; Choinzonov, E. L.

    2016-08-01

    Progression of tumors and metastasis in particular is one of the main reasons of the high mortality rate among cancer patients. The primary role in developing metastases plays cell locomotion which requires remodeling of the actin cytoskeleton. Form, dynamics, localization and mechanical properties of the actin cytoskeleton are regulated by a variety of actin-binding proteins, which include the adenylyl cyclase-associated protein 1 (CAP1). The study is devoted to the investigation of CAP1 level depending on the presence or absence of metastases in patients with squamous cell carcinoma of the head and neck (SCCHN) and non-small cell lung cancer (NSCLC). The results show the contribution of CAP1 to SCCHN and NSCLC progression. We detected the connection between the tissue protein CAP1 level and the stage of NSCLC and SCCHN disease. Also the levels of the CAP1 protein in tissues of primary tumors and metastases in lung cancer were different. Our data showed that CAP is important in the development of metastases, which suggests further perspectives in the study of this protein for projecting metastasis of NSCLC and SCCHN.

  1. Adenylyl cyclase A expression is tip-specific in Dictyostelium slugs and directs StatA nuclear translocation and CudA gene expression.

    PubMed

    Verkerke-van Wijk, I; Fukuzawa, M; Devreotes, P N; Schaap, P

    2001-06-01

    cAMP oscillations, generated by adenylyl cyclase A (ACA), coordinate cell aggregation in Dictyostelium and have also been implicated in organizer function during multicellular development. We used a gene fusion of the ACA promoter with a labile lacZ derivative to study the expression pattern of ACA. During aggregation, most cells expressed ACA, but thereafter expression was lost in all cells except those of the anterior tip. Before aggregation, ACA transcription was strongly upregulated by nanomolar cAMP pulses. Postaggregative transcription was sustained by nanomolar cAMP pulses, but downregulated by a continuous micromolar cAMP stimulus and by the stalk-cell-inducing factor DIF. Earlier work showed that the transcription factor StatA displays tip-specific nuclear translocation and directs tip-specific expression of the nuclear protein CudA, which is essential for culmination. Both StatA and CudA were present in nuclei throughout the entire slug in an aca null mutant that expresses ACA from the constitutive actin15 promoter. This suggests that the tip-specific expression of ACA directs tip-specific nuclear translocation of StatA and tip-specific expression of CudA. Copyright 2001 Academic Press.

  2. [THE CHANGES OF NOCICEPTIVE THRESHOLD AND ACTIVITY OF THE ADENYLYL CYCLASE SYSTEM IN THE SKELETAL MUSCLES OF RATS WITH ACUTE AND MILD TYPE 1 DIABETES MELLITUS ].

    PubMed

    Shipilov, V N; Trost, A M; Chistyakova, O V; Derkach, K V; Shpakov, A O

    2016-02-01

    Diabetic peripheral neuropathy (DPN) is one of the most common complications of the type 1 diabetes mellitus (DM1). The aim of the work was to study the dynamics of a painful DPN and functional state of the hormone-sensitive ACSS in the skeletal muscles of rats with the models of acute and mild DM1, as well as the study of impact on them of insulin therapy with different ways of hormone delivery - intranasal and peripheral. In both models of DM1, the level of nociceptive threshold in rats decreased and the stimulatory effects of guanine nucleotides (GppNHp) and adrenergic agonists (isoproterenol, BRL-37344) on adenylyl cyclase (AC) activity were attenuated. The AC stimulating effect of relaxin decreased in animals with acute DM1, but in mild DM1, the decrease was insignificant. Peripheral administration of insulin in rats with acute DM1 increased the nociceptive threshold and partially restored the AC effect of ß 3-agonist BRL-37344. Intranasal administration of insulin in rats with DM1 also increased the nociceptive threshold and partially restored the basal and BRL-37344-stimulated AC activity in the skeletal muscles of diabetic animals. Thus, in the skeletal muscles of rats with acute and mild DM1 the nociceptive sensitivity and the functions of ACSS were disturbed, and they were partially restored by the treatment with peripheral (acute DM1) or intranasal (mild DM1) insulin.

  3. Differential profile of typical, atypical and third generation antipsychotics at human 5-HT7a receptors coupled to adenylyl cyclase: detection of agonist and inverse agonist properties.

    PubMed

    Rauly-Lestienne, Isabelle; Boutet-Robinet, Elisa; Ailhaud, Marie-Christine; Newman-Tancredi, Adrian; Cussac, Didier

    2007-10-01

    5-HT(7) receptors are present in thalamus and limbic structures, and a possible role of these receptors in the pathology of schizophrenia has been evoked. In this study, we examined binding affinity and agonist/antagonist/inverse agonist properties at these receptors of a large series of antipsychotics, i.e., typical, atypical, and third generation compounds preferentially targeting D(2) and 5-HT(1A) sites. Adenylyl cyclase (AC) activity was measured in HEK293 cells stably expressing the human (h) 5-HT(7a) receptor isoform. 5-HT and 5-CT increased cyclic adenosine monophosphate level by about 20-fold whereas (+)-8-OH-DPAT, the antidyskinetic agent sarizotan, and the novel antipsychotic compound bifeprunox exhibited partial agonist properties at h5-HT(7a) receptors stimulating AC. Other compounds antagonized 5-HT-induced AC activity with pK (B) values which correlated with their pK (i) as determined by competition binding vs [(3)H]5-CT. The selective 5-HT(7) receptor ligand, SB269970, was the most potent antagonist. For antipsychotic compounds, the following rank order of antagonism potency (pK (B)) was ziprasidone > tiospirone > SSR181507 > or = clozapine > or = olanzapine > SLV-314 > SLV-313 > or = aripiprazole > or = chlorpromazine > nemonapride > haloperidol. Interestingly, pretreatment of HEK293-h5-HT(7a) cells with forskolin enhanced basal AC activity and revealed inverse agonist properties for both typical and atypical antipsychotics as well as for aripiprazole. In contrast, other novel antipsychotics exhibited diverse 5-HT(7a) properties; SLV-313 and SLV-314 behaved as quasi-neutral antagonists, SSR181507 acted as an inverse agonist, and bifeprunox as a partial agonist, as mentioned above. In conclusion, the differential properties of third generation antipsychotics at 5-HT(7) receptors may influence their antipsychotic profile.

  4. Overexpression of Adenylyl Cyclase Encoded by the Mycobacterium tuberculosis Rv2212 Gene Confers Improved Fitness, Accelerated Recovery from Dormancy and Enhanced Virulence in Mice.

    PubMed

    Shleeva, Margarita O; Kondratieva, Tatyana K; Demina, Galina R; Rubakova, Elvira I; Goncharenko, Anna V; Apt, Alexander S; Kaprelyants, Arseny S

    2017-01-01

    Earlier we demonstrated that the adenylyl cyclase (AC) encoded by the MSMEG_4279 gene plays a key role in the resuscitation and growth of dormant Mycobacterium smegmatis and that overexpression of this gene leads to an increase in intracellular cAMP concentration and prevents the transition of M. smegmatis from active growth to dormancy in an extended stationary phase accompanied by medium acidification. We surmised that the homologous Rv2212 gene of M. tuberculosis ( Mtb ), the main cAMP producer, plays similar physiological roles by supporting, under these conditions, the active state and reactivation of dormant bacteria. To test this hypothesis, we established Mtb strain overexpressing Rv2212 and compared its in vitro and in vivo growth characteristics with a control strain. In vitro , the AC-overexpressing pMind Rv2212 strain demonstrated faster growth in a liquid medium, prolonged capacity to form CFUs and a significant delay or even prevention of transition toward dormancy. AC-overexpressing cells exhibited easier recovery from dormancy. In vivo , AC-overexpressing bacteria demonstrated significantly higher growth rates (virulence) in the lungs and spleens of infected mice compared to the control strain, and, unlike the latter, killed mice in the TB-resistant strain before month 8 of infection. Even in the absence of selecting hygromycin B, all pMind Rv2212 CFUs retained the Rv2212 insert during in vivo growth, strongly suggesting that AC overexpression is beneficial for bacteria. Taken together, our results indicate that cAMP supports the maintenance of Mtb cells vitality under unfavorable conditions in vitro and their virulence in vivo .

  5. Chronic treatment with escitalopram but not R-citalopram translocates Galpha(s) from lipid raft domains and potentiates adenylyl cyclase: a 5-hydroxytryptamine transporter-independent action of this antidepressant compound.

    PubMed

    Zhang, Lanqiu; Rasenick, Mark M

    2010-03-01

    Chronic antidepressant treatment has been shown to increase adenylyl cyclase activity, in part, due to translocation of Galpha(s) from lipid rafts to a nonraft fraction of the plasma membrane where they engage in a more facile stimulation of adenylyl cyclase. This effect holds for multiple classes of antidepressants, and for serotonin uptake inhibitors, it occurs in the absence of the serotonin transporter. In the present study, we examined the change in the amount of Galpha(s) in lipid raft and whole cell lysate after exposing C6 cells to escitalopram. The results showed that chronic (but not acute) escitalopram decreased the content of Galpha(s) in lipid rafts, whereas there was no change in overall Galpha(s) content. These effects were drug dose- and exposure time-dependent. Although R-citalopram has been reported to antagonize some effects of escitalopram, this compound was without effect on Galpha(s) localization in lipid rafts, and R-citalopram did not inhibit these actions of escitalopram. Escitalopram treatment increased cAMP accumulation, and this seemed due to increased coupling between Galpha(s) and adenylyl cyclase. Thus, escitalopram is potent, rapid and efficacious in translocating Galpha(s) from lipid rafts, and this effect seems to occur independently of 5-hydroxytryptamine transporters. Our results suggest that, although antidepressants display distinct affinities for well identified targets (e.g., monoamine transporters), several presynaptic and postsynaptic molecules are probably modified during chronic antidepressant treatment, and these additional targets may be required for clinical efficacy of these drugs.

  6. Chronic Treatment with Escitalopram but Not R-Citalopram Translocates Gαs from Lipid Raft Domains and Potentiates Adenylyl Cyclase: A 5-Hydroxytryptamine Transporter-Independent Action of This Antidepressant Compound

    PubMed Central

    Zhang, Lanqiu

    2010-01-01

    Chronic antidepressant treatment has been shown to increase adenylyl cyclase activity, in part, due to translocation of Gαs from lipid rafts to a nonraft fraction of the plasma membrane where they engage in a more facile stimulation of adenylyl cyclase. This effect holds for multiple classes of antidepressants, and for serotonin uptake inhibitors, it occurs in the absence of the serotonin transporter. In the present study, we examined the change in the amount of Gαs in lipid raft and whole cell lysate after exposing C6 cells to escitalopram. The results showed that chronic (but not acute) escitalopram decreased the content of Gαs in lipid rafts, whereas there was no change in overall Gαs content. These effects were drug dose- and exposure time-dependent. Although R-citalopram has been reported to antagonize some effects of escitalopram, this compound was without effect on Gαs localization in lipid rafts, and R-citalopram did not inhibit these actions of escitalopram. Escitalopram treatment increased cAMP accumulation, and this seemed due to increased coupling between Gαs and adenylyl cyclase. Thus, escitalopram is potent, rapid and efficacious in translocating Gαs from lipid rafts, and this effect seems to occur independently of 5-hydroxytryptamine transporters. Our results suggest that, although antidepressants display distinct affinities for well identified targets (e.g., monoamine transporters), several presynaptic and postsynaptic molecules are probably modified during chronic antidepressant treatment, and these additional targets may be required for clinical efficacy of these drugs. PMID:19996298

  7. Integrative Signaling Networks of Membrane Guanylate Cyclases: Biochemistry and Physiology

    PubMed Central

    Sharma, Rameshwar K.; Duda, Teresa; Makino, Clint L.

    2016-01-01

    This monograph presents a historical perspective of cornerstone developments on the biochemistry and physiology of mammalian membrane guanylate cyclases (MGCs), highlighting contributions made by the authors and their collaborators. Upon resolution of early contentious studies, cyclic GMP emerged alongside cyclic AMP, as an important intracellular second messenger for hormonal signaling. However, the two signaling pathways differ in significant ways. In the cyclic AMP pathway, hormone binding to a G protein coupled receptor leads to stimulation or inhibition of an adenylate cyclase, whereas the cyclic GMP pathway dispenses with intermediaries; hormone binds to an MGC to affect its activity. Although the cyclic GMP pathway is direct, it is by no means simple. The modular design of the molecule incorporates regulation by ATP binding and phosphorylation. MGCs can form complexes with Ca2+-sensing subunits that either increase or decrease cyclic GMP synthesis, depending on subunit identity. In some systems, co-expression of two Ca2+ sensors, GCAP1 and S100B with ROS-GC1 confers bimodal signaling marked by increases in cyclic GMP synthesis when intracellular Ca2+ concentration rises or falls. Some MGCs monitor or are modulated by carbon dioxide via its conversion to bicarbonate. One MGC even functions as a thermosensor as well as a chemosensor; activity reaches a maximum with a mild drop in temperature. The complexity afforded by these multiple limbs of operation enables MGC networks to perform transductions traditionally reserved for G protein coupled receptors and Transient Receptor Potential (TRP) ion channels and to serve a diverse array of functions, including control over cardiac vasculature, smooth muscle relaxation, blood pressure regulation, cellular growth, sensory transductions, neural plasticity and memory. PMID:27695398

  8. Discovery of G protein signaling.

    PubMed

    Selinger, Zvi

    2008-01-01

    The mechanism of transmembrane signaling by the receptor-activated adenylyl cyclase was an enigma. It was suggested that hydrolysis of GTP is a turn-off mechanism that resets the active adenylyl cyclase to the inactive state. To test this hypothesis, we developed a specific GTPase assay and found that the catecholamine adrenergic agonists stimulated the hydrolysis of GTP. To resolve the question of how the hormone concurrently stimulates GTP hydrolysis and activates the adenylyl cyclase, we suggested the regulatory GTPase cycle. Thus, because the hormone facilitates the binding of GTP, which is subsequently hydrolyzed, the regulatory cycle results in a hormone-stimulated GTPase activity. This model also predicts that two mechanisms could account for stimulation of adenylyl cyclase activity-either by the familiar hormone stimulation of the activation reaction or by an inhibition of the turn-off reaction. Indeed, we showed that cholera toxin enhances adenylyl cyclase activity by inhibition of GTP hydrolysis. Finally, we also showed that the hormone-activated receptor stimulates adenylyl cyclase activity by facilitating the exchange of bound GDP for free GTP. Thus, we presented, for the first time, an explicit mechanism for receptor action.

  9. Membrane guanylate cyclase is a beautiful signal transduction machine: overview.

    PubMed

    Sharma, Rameshwar K

    2010-01-01

    This article is a sequel to the four earlier comprehensive reviews which covered the field of membrane guanylate cyclase from its origin to the year 2002 (Sharma in Mol Cell Biochem 230:3-30, 2002) and then to the year 2004 (Duda et al. in Peptides 26:969-984, 2005); and of the Ca(2+)-modulated membrane guanylate cyclase to the year 1997 (Pugh et al. in Biosci Rep 17:429-473, 1997) and then to 2004 (Sharma et al. in Curr Top Biochem Res 6:111-144, 2004). This article contains three parts. The first part is "Historical"; it is brief, general, and freely borrowed from the earlier reviews, covering the field from its origin to the year 2004 (Sharma in Mol Cell Biochem, 230:3-30, 2002; Duda et al. in Peptides 26:969-984, 2005). The second part focuses on the "Ca(2+)-modulated ROS-GC membrane guanylate cyclase subfamily". It is divided into two sections. Section "Historical" and covers the area from its inception to the year 2004. It is also freely borrowed from an earlier review (Sharma et al. in Curr Top Biochem Res 6:111-144, 2004). Section "Ca(2+)-modulated ROS-GC membrane guanylate cyclase subfamily" covers the area from the year 2004 to May 2009. The objective is to focus on the chronological development, recognize major contributions of the original investigators, correct misplaced facts, and project on the future trend of the field of mammalian membrane guanylate cyclase. The third portion covers the present status and concludes with future directions in the field.

  10. Continuous activation of pituitary adenylate cyclase-activating polypeptide receptors elicits antipodal effects on cyclic AMP and inositol phospholipid signaling pathways in CATH.a cells: role of protein synthesis and protein kinases.

    PubMed

    Muller, A; Lutz-Bucher, B; Kienlen-Campard, P; Koch, B; Loeffler, J P

    1998-04-01

    Continuous exposure of cells to agonists develops a process that determines the extent to which the cells eventually respond to further stimuli. Here we used CATH.a cells (a catecholaminergic neuron-like cell line), which express pituitary adenylate cyclase-activating polypeptide (PACAP) receptors linked to both adenylyl cyclase and phospholipase C-beta pathways, to investigate the influence of prolonged hormonal treatment on dual signaling and gene transcription. Prolonged incubation of cells with PACAP failed to down-regulate the density and affinity of membrane binding sites and caused opposite changes in messenger systems: PACAP-stimulated cyclic AMP accumulation was attenuated in a time- and dose-dependent fashion (t(1/2) = 6.7 h and IC50 = 0.1 nM), whereas phosphoinositide turnover was overstimulated. Both effects were insensitive to pertussis toxin, whereas the drop in cyclic AMP concentration was also unchanged in the presence of 3-isobutyl-1-methylxanthine, indicating that neither Gi-like proteins nor cyclic nucleotide phosphodiesterases play a critical role in these processes. Blockade of protein synthesis with cycloheximide, as well as inhibition by H89 of cyclic AMP-dependent protein kinase (but not by bisindolylmaleimide of protein kinase C) antagonized the influences exerted by PACAP on adenylyl cyclase activity and inositol phosphate formation. Transcription of the chimeric GAL4-CREB construct, transiently transfected into CATH.a cells, was stimulated by PACAP, and this effect was potentiated as a result of chronic PACAP treatment. The results of the present investigation provide new insight into the possible differential regulation and cross-talks of transduction signals of receptors linked to multiplex signaling. They demonstrate that prolonged exposure of CATH.a cells to PACAP results in the desensitization of the cyclic AMP pathway and superinduction of the inositol phosphate signal, through protein neosynthesis and cyclic AMP-dependent protein

  11. High density and ligand affinity confer ultrasensitive signal detection by a guanylyl cyclase chemoreceptor

    PubMed Central

    Pichlo, Magdalena; Bungert-Plümke, Stefanie; Weyand, Ingo; Seifert, Reinhard; Bönigk, Wolfgang; Strünker, Timo; Kashikar, Nachiket Dilip; Goodwin, Normann; Müller, Astrid; Körschen, Heinz G.; Collienne, Ursel; Pelzer, Patric; Van, Qui; Enderlein, Jörg; Klemm, Clementine; Krause, Eberhard; Trötschel, Christian; Poetsch, Ansgar; Kremmer, Elisabeth

    2014-01-01

    Guanylyl cyclases (GCs), which synthesize the messenger cyclic guanosine 3′,5′-monophosphate, control several sensory functions, such as phototransduction, chemosensation, and thermosensation, in many species from worms to mammals. The GC chemoreceptor in sea urchin sperm can decode chemoattractant concentrations with single-molecule sensitivity. The molecular and cellular underpinnings of such ultrasensitivity are not known for any eukaryotic chemoreceptor. In this paper, we show that an exquisitely high density of 3 × 105 GC chemoreceptors and subnanomolar ligand affinity provide a high ligand-capture efficacy and render sperm perfect absorbers. The GC activity is terminated within 150 ms by dephosphorylation steps of the receptor, which provides a means for precise control of the GC lifetime and which reduces “molecule noise.” Compared with other ultrasensitive sensory systems, the 10-fold signal amplification by the GC receptor is surprisingly low. The hallmarks of this signaling mechanism provide a blueprint for chemical sensing in small compartments, such as olfactory cilia, insect antennae, or even synaptic boutons. PMID:25135936

  12. Exploratory behaviour in NO-dependent cyclase mutants of Drosophila shows defects in coincident neuronal signalling

    PubMed Central

    Tinette, Sylvette; Zhang, Lixing; Garnier, Amélie; Engler, Gilbert; Tares, Sophie; Robichon, Alain

    2007-01-01

    Background Drosophila flies explore the environment very efficiently in order to colonize it. They explore collectively, not individually, so that when a few land on a food spot, they attract the others by signs. This behaviour leads to aggregation of individuals and optimizes the screening of mates and egg-laying on the most favourable food spots. Results Flies perform cycles of exploration/aggregation depending on the resources of the environment. This behavioural ecology constitutes an excellent model for analyzing simultaneous processing of neurosensory information. We reasoned that the decision of flies to land somewhere in order to achieve aggregation is based on simultaneous integration of signals (visual, olfactory, acoustic) during their flight. On the basis of what flies do in nature, we designed laboratory tests to analyze the phenomenon of neuronal coincidence. We screened many mutants of genes involved in neuronal metabolism and the synaptic machinery. Conclusion Mutants of NO-dependent cyclase show a specifically-marked behaviour phenotype, but on the other hand they are associated with moderate biochemical defects. We show that these mutants present errors in integrative and/or coincident processing of signals, which are not reducible to the functions of the peripheral sensory cells. PMID:17683617

  13. Long-range allosteric signaling in red light–regulated diguanylyl cyclases

    PubMed Central

    Gourinchas, Geoffrey; Etzl, Stefan; Göbl, Christoph; Vide, Uršula; Madl, Tobias; Winkler, Andreas

    2017-01-01

    Nature has evolved an astonishingly modular architecture of covalently linked protein domains with diverse functionalities to enable complex cellular networks that are critical for cell survival. The coupling of sensory modules with enzymatic effectors allows direct allosteric regulation of cellular signaling molecules in response to diverse stimuli. We present molecular details of red light–sensing bacteriophytochromes linked to cyclic dimeric guanosine monophosphate–producing diguanylyl cyclases. Elucidation of the first crystal structure of a full-length phytochrome with its enzymatic effector, in combination with the characterization of light-induced changes in conformational dynamics, reveals how allosteric light regulation is fine-tuned by the architecture and composition of the coiled-coil sensor-effector linker and also the central helical spine. We anticipate that consideration of molecular principles of sensor-effector coupling, going beyond the length of the characteristic linker, and the appreciation of dynamically driven allostery will open up new directions for the design of novel red light–regulated optogenetic tools. PMID:28275738

  14. ATRIAL NATRIURETIC FACTOR RECEPTOR GUANYLATE CYCLASE SIGNALING: NEW ATP- REGULATED TRANSDUCTION MOTIF

    PubMed Central

    Duda, Teresa; Bharill, Shashank; Wojtas, Ireneusz; Yadav, Prem; Gryczynski, Ignacy; Gryczynski, Zygmunt; Sharma, Rameshwar K.

    2010-01-01

    ANF-RGC$ membrane guanylate cyclase is the receptor for the hypotensive peptide hormones, atrial natriuretic factor (ANF) and type B natriuretic peptide (BNP). It is a single transmembrane spanning protein. Binding the hormone to the extracellular domain activates its intracellular catalytic domain. This results in accelerated production of cyclic GMP, a second messenger in controlling blood pressure, cardiac vasculature and fluid secretion. ATP is the obligatory transducer of the ANF signal. It works through its ATP regulated module, ARM, which is juxtaposed to the C-terminal side of the transmembrane domain. Upon interaction, ATP induces a cascade of temporal and spatial changes in the ARM, which, finally, result in activation of the catalytic module. Although the exact nature and the details of these changes are not known, some of these have been stereographed in the simulated three-dimensional model of the ARM and validated biochemically. Through comprehensive techniques ofsteady-state, time-resolved tryptophan fluorescence and Forster Resonance Energy Transfer (FRET), site-directed and deletion-mutagenesis, and reconstitution, the present study validates and explains themechanism of the model-based predicted transduction role of the ARM’s structural motif, 669WTAPELL675. This motif is critical in the ATP-dependent ANF signaling. Molecular modeling shows that ATP binding exposes the 669WTAPELL675 motif, the exposure, in turn, facilitates its interaction and activation of the catalytic module. These principles of the model have been experimentally validated. This knowledge brings us a step closer to our understanding of the mechanism by which the ATP-dependent spatial changes within the ARM cause ANF signaling of ANF-RGC. PMID:19137266

  15. Sustained signalling by PTH modulates IP3 accumulation and IP3 receptors through cyclic AMP junctions

    PubMed Central

    Meena, Abha; Tovey, Stephen C.; Taylor, Colin W.

    2015-01-01

    ABSTRACT Parathyroid hormone (PTH) stimulates adenylyl cyclase through type 1 PTH receptors (PTH1R) and potentiates the Ca2+ signals evoked by carbachol, which stimulates formation of inositol 1,4,5-trisphosphate (IP3). We confirmed that in HEK cells expressing PTH1R, acute stimulation with PTH(1-34) potentiated carbachol-evoked Ca2+ release. This was mediated by locally delivered cyclic AMP (cAMP), but unaffected by inhibition of protein kinase A (PKA), exchange proteins activated by cAMP, cAMP phosphodiesterases (PDEs) or substantial inhibition of adenylyl cyclase. Sustained stimulation with PTH(1-34) causes internalization of PTH1R–adenylyl cyclase signalling complexes, but the consequences for delivery of cAMP to IP3R within cAMP signalling junctions are unknown. Here, we show that sustained stimulation with PTH(1-34) or with PTH analogues that do not evoke receptor internalization reduced the potentiated Ca2+ signals and attenuated carbachol-evoked increases in cytosolic IP3. Similar results were obtained after sustained stimulation with NKH477 to directly activate adenylyl cyclase, or with the membrane-permeant analogue of cAMP, 8-Br-cAMP. These responses were independent of PKA and unaffected by substantial inhibition of adenylyl cyclase. During prolonged stimulation with PTH(1-34), hyperactive cAMP signalling junctions, within which cAMP is delivered directly and at saturating concentrations to its targets, mediate sensitization of IP3R and a more slowly developing inhibition of IP3 accumulation. PMID:25431134

  16. Pituitary adenylate cyclase 1 receptor internalization and endosomal signaling mediate the pituitary adenylate cyclase activating polypeptide-induced increase in guinea pig cardiac neuron excitability.

    PubMed

    Merriam, Laura A; Baran, Caitlin N; Girard, Beatrice M; Hardwick, Jean C; May, Victor; Parsons, Rodney L

    2013-03-06

    After G-protein-coupled receptor activation and signaling at the plasma membrane, the receptor complex is often rapidly internalized via endocytic vesicles for trafficking into various intracellular compartments and pathways. The formation of signaling endosomes is recognized as a mechanism that produces sustained intracellular signals that may be distinct from those generated at the cell surface for cellular responses including growth, differentiation, and survival. Pituitary adenylate cyclase activating polypeptide (PACAP; Adcyap1) is a potent neurotransmitter/neurotrophic peptide and mediates its diverse cellular functions in part through internalization of its cognate G-protein-coupled PAC1 receptor (PAC1R; Adcyap1r1). In the present study, we examined whether PAC1R endocytosis participates in the regulation of neuronal excitability. Although PACAP increased excitability in 90% of guinea pig cardiac neurons, pretreatment with Pitstop 2 or dynasore to inhibit clathrin and dynamin I/II, respectively, suppressed the PACAP effect. Subsequent addition of inhibitor after the PACAP-induced increase in excitability developed gradually attenuated excitability with no changes in action potential properties. Likewise, the PACAP-induced increase in excitability was markedly decreased at ambient temperature. Receptor trafficking studies with GFP-PAC1 cell lines demonstrated the efficacy of Pitstop 2, dynasore, and low temperatures at suppressing PAC1R endocytosis. In contrast, brefeldin A pretreatments to disrupt Golgi vesicle trafficking did not blunt the PACAP effect, and PACAP/PAC1R signaling still increased neuronal cAMP production even with endocytic blockade. Our results demonstrate that PACAP/PAC1R complex endocytosis is a key step for the PACAP modulation of cardiac neuron excitability.

  17. Corruption of homeostatic mechanisms in the guanylyl cyclase C signaling pathway underlying colorectal tumorigenesis

    PubMed Central

    Waldman, Scott A

    2010-01-01

    Colon cancer, the second leading cause of cancer-related mortality worldwide, originates from the malignant transformation of intestinal epithelial cells. The intestinal epithelium undergoes a highly organized process of rapid regeneration along the crypt-villus axis, characterized by proliferation, migration, differentiation and apoptosis, whose coordination is essential to maintaining the mucosal barrier. Disruption of these homeostatic processes predisposes cells to mutations in tumor suppressors or oncogenes, whose dysfunction provides transformed cells an evolutionary growth advantage. While sequences of genetic mutations at different stages along the neoplastic continuum have been established, little is known of the events initiating tumorigenesis prior to adenomatous polyposis coli (APC) mutations. Here, we examine a role for the corruption of homeostasis induced by silencing novel tumor suppressors, including the intestine-specific transcription factor CDX2 and its gene target guanylyl cyclase C (GCC), as early events predisposing cells to mutations in APC and other sequential genes that initiate colorectal cancer. CDX2 and GCC maintain homeostatic regeneration in the intestine by restricting cell proliferation, promoting cell maturation and adhesion, regulating cell migration and defending the intestinal barrier and genomic integrity. Elimination of CDX2 or GCC promotes intestinal tumor initiation and growth in aged mice, mice carrying APC mutations or mice exposed to carcinogens. The roles of CDX2 and GCC in suppressing intestinal tumorigenesis, universal disruption in their signaling through silencing of hormones driving GCC, and the uniform overexpression of GCC by tumors underscore the potential value of oral replacement with GCC ligands as targeted prevention and therapy for colorectal cancer. PMID:20592492

  18. sAC as a model for understanding the impact of endosymbiosis on cell signaling.

    PubMed

    Blackstone, Neil W

    2014-12-01

    As signaling pathways evolve, selection for new functions guides the co-option of existing material. Major transitions in the history of life, including the evolution of eukaryotes and multicellularity, exemplify this process. These transitions provided both strong selection and a plenitude of available material for the evolution of signaling pathways. Mechanisms that evolved to mediate conflict during the evolution of eukaryotes may subsequently have been co-opted during the many independent derivations of multicellularity. The soluble adenylyl cyclase (sAC) signaling pathway illustrates this hypothesis. Class III adenylyl cyclases, which include sAC, are found in bacteria, including the α-proteobacteria. These adenylyl cyclases are the only ones present in eukaryotes but appear to be absent in archaeans. This pattern suggests that the mitochondrial endosymbiosis brought sAC signaling to eukaryotes as part of an intact module. After transfer to the proto-nuclear genome, this module was then co-opted into numerous new functions. In the evolution of eukaryotes, sAC signaling may have mediated conflicts by maintaining metabolic homeostasis. In the evolution of multicellularity, in different lineages sAC may have been co-opted into parallel tasks originally related to conflict mediation. Elucidating the history of the sAC pathway may be relatively straightforward because it is ubiquitous and linked to near universal metabolic by-products (CO₂/HCO(3)(-)). Other signaling pathways (e.g., those involving STAT and VEGF) present a greater challenge but may suggest a complementary pattern. The impact of the mitochondrial endosymbiosis on cell signaling may thus have been profound. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. The Circadian Neuropeptide PDF Signals Preferentially through a Specific Adenylate Cyclase Isoform AC3 in M Pacemakers of Drosophila

    PubMed Central

    Duvall, Laura B.; Taghert, Paul H.

    2012-01-01

    The neuropeptide Pigment Dispersing Factor (PDF) is essential for normal circadian function in Drosophila. It synchronizes the phases of M pacemakers, while in E pacemakers it decelerates their cycling and supports their amplitude. The PDF receptor (PDF-R) is present in both M and subsets of E cells. Activation of PDF-R stimulates cAMP increases in vitro and in M cells in vivo. The present study asks: What is the identity of downstream signaling components that are associated with PDF receptor in specific circadian pacemaker neurons? Using live imaging of intact fly brains and transgenic RNAi, we show that adenylate cyclase AC3 underlies PDF signaling in M cells. Genetic disruptions of AC3 specifically disrupt PDF responses: they do not affect other Gs-coupled GPCR signaling in M cells, they can be rescued, and they do not represent developmental alterations. Knockdown of the Drosophila AKAP-like scaffolding protein Nervy also reduces PDF responses. Flies with AC3 alterations show behavioral syndromes consistent with known roles of M pacemakers as mediated by PDF. Surprisingly, disruption of AC3 does not alter PDF responses in E cells—the PDF-R(+) LNd. Within M pacemakers, PDF-R couples preferentially to a single AC, but PDF-R association with a different AC(s) is needed to explain PDF signaling in the E pacemakers. Thus critical pathways of circadian synchronization are mediated by highly specific second messenger components. These findings support a hypothesis that PDF signaling components within target cells are sequestered into “circadian signalosomes,” whose compositions differ between E and M pacemaker cell types. PMID:22679392

  20. The circadian neuropeptide PDF signals preferentially through a specific adenylate cyclase isoform AC3 in M pacemakers of Drosophila.

    PubMed

    Duvall, Laura B; Taghert, Paul H

    2012-01-01

    The neuropeptide Pigment Dispersing Factor (PDF) is essential for normal circadian function in Drosophila. It synchronizes the phases of M pacemakers, while in E pacemakers it decelerates their cycling and supports their amplitude. The PDF receptor (PDF-R) is present in both M and subsets of E cells. Activation of PDF-R stimulates cAMP increases in vitro and in M cells in vivo. The present study asks: What is the identity of downstream signaling components that are associated with PDF receptor in specific circadian pacemaker neurons? Using live imaging of intact fly brains and transgenic RNAi, we show that adenylate cyclase AC3 underlies PDF signaling in M cells. Genetic disruptions of AC3 specifically disrupt PDF responses: they do not affect other Gs-coupled GPCR signaling in M cells, they can be rescued, and they do not represent developmental alterations. Knockdown of the Drosophila AKAP-like scaffolding protein Nervy also reduces PDF responses. Flies with AC3 alterations show behavioral syndromes consistent with known roles of M pacemakers as mediated by PDF. Surprisingly, disruption of AC3 does not alter PDF responses in E cells--the PDF-R(+) LNd. Within M pacemakers, PDF-R couples preferentially to a single AC, but PDF-R association with a different AC(s) is needed to explain PDF signaling in the E pacemakers. Thus critical pathways of circadian synchronization are mediated by highly specific second messenger components. These findings support a hypothesis that PDF signaling components within target cells are sequestered into "circadian signalosomes," whose compositions differ between E and M pacemaker cell types.

  1. Pituitary adenylate cyclase activating polypeptide and PAC1 receptor signaling increase Homer 1a expression in central and peripheral neurons.

    PubMed

    Girard, Beatrice M; Keller, Emily T; Schutz, Kristin C; May, Victor; Braas, Karen M

    2004-12-15

    Pituitary adenylate cyclase activating polypeptides (PACAP) and PAC1 receptor signaling have diverse roles in central and peripheral nervous system development and function. In recent microarray analyses for PACAP and PAC1 receptor modulation of neuronal transcripts, the mRNA of Homer 1a (H1a), which encodes the noncrosslinking and immediate early gene product isoform of Homer, was identified to be strongly upregulated in superior cervical ganglion (SCG) sympathetic neurons. Given the prominent roles of Homer in synaptogenesis, synaptic protein complex assembly and receptor/channel signaling, we have examined the ability for PACAP to induce H1a expression in sympathetic, cortical and hippocampal neurons to evaluate more comprehensively the roles of PACAP in synaptic function. In both central and peripheral neuronal cultures, PACAP peptides increased transiently H1a transcript levels approximately 3.5- to 6-fold. From real-time quantitative PCR measurements, the temporal patterns of PACAP-mediated H1a mRNA induction among the different neuronal cultures appeared similar although the onset of sympathetic H1a transcript expression appeared protracted. The increase in H1a transcripts was accompanied by increases in H1a protein levels. Comparative studies with VIP and PACAP(6-38) antagonist demonstrated that the PACAP effects reflected PAC1 receptor activation and signaling. The PAC1 receptor isoforms expressed in central and peripheral neurons can engage diverse intracellular second messenger systems, and studies using selective signaling pathway inhibitors demonstrated that the cyclic AMP/PKA and MEK/ERK cascades are principal mediators of the PACAP-mediated H1a induction response. In modulating H1a transcript and protein expression, these studies may implicate broad roles for PACAP and PAC1 receptor signaling in synaptic development and plasticity.

  2. Clathrin-dependent internalization, signaling, and metabolic processing of guanylyl cyclase/natriuretic peptide receptor-A.

    PubMed

    Somanna, Naveen K; Mani, Indra; Tripathi, Satyabha; Pandey, Kailash N

    2018-04-01

    Cardiac hormones, atrial and brain natriuretic peptides (ANP and BNP), have pivotal roles in renal hemodynamics, neuroendocrine signaling, blood pressure regulation, and cardiovascular homeostasis. Binding of ANP and BNP to the guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) induces rapid internalization and trafficking of the receptor via endolysosomal compartments, with concurrent generation of cGMP. However, the mechanisms of the endocytotic processes of NPRA are not well understood. The present study, using 125 I-ANP binding assay and confocal microscopy, examined the function of dynamin in the internalization of NPRA in stably transfected human embryonic kidney-293 (HEK-293) cells. Treatment of recombinant HEK-293 cells with ANP time-dependently accelerated the internalization of receptor from the cell surface to the cell interior. However, the internalization of ligand-receptor complexes of NPRA was drastically decreased by the specific inhibitors of clathrin- and dynamin-dependent receptor internalization, almost 85% by monodansylcadaverine, 80% by chlorpromazine, and 90% by mutant dynamin, which are specific blockers of endocytic vesicle formation. Visualizing the internalization of NPRA and enhanced GFP-tagged NPRA in HEK-293 cells by confocal microscopy demonstrated the formation of endocytic vesicles after 5 min of ANP treatment; this effect was blocked by the inhibitors of clathrin and by mutant dynamin construct. Our results suggest that NPRA undergoes internalization via clathrin-mediated endocytosis as part of its normal itinerary, including trafficking, signaling, and metabolic degradation.

  3. A cardiac pathway of cyclic GMP-independent signaling of guanylyl cyclase A, the receptor for atrial natriuretic peptide

    PubMed Central

    Klaiber, Michael; Dankworth, Beatrice; Kruse, Martin; Hartmann, Michael; Nikolaev, Viacheslav O.; Yang, Ruey-Bing; Völker, Katharina; Gaßner, Birgit; Oberwinkler, Heike; Feil, Robert; Freichel, Marc; Groschner, Klaus; Skryabin, Boris V.; Frantz, Stefan; Birnbaumer, Lutz; Pongs, Olaf; Kuhn, Michaela

    2011-01-01

    Cardiac atrial natriuretic peptide (ANP) regulates arterial blood pressure, moderates cardiomyocyte growth, and stimulates angiogenesis and metabolism. ANP binds to the transmembrane guanylyl cyclase (GC) receptor, GC-A, to exert its diverse functions. This process involves a cGMP-dependent signaling pathway preventing pathological [Ca2+]i increases in myocytes. In chronic cardiac hypertrophy, however, ANP levels are markedly increased and GC-A/cGMP responses to ANP are blunted due to receptor desensitization. Here we show that, in this situation, ANP binding to GC-A stimulates a unique cGMP-independent signaling pathway in cardiac myocytes, resulting in pathologically elevated intracellular Ca2+ levels. This pathway involves the activation of Ca2+‐permeable transient receptor potential canonical 3/6 (TRPC3/C6) cation channels by GC-A, which forms a stable complex with TRPC3/C6 channels. Our results indicate that the resulting cation influx activates voltage-dependent L-type Ca2+ channels and ultimately increases myocyte Ca2+i levels. These observations reveal a dual role of the ANP/GC-A–signaling pathway in the regulation of cardiac myocyte Ca2+i homeostasis. Under physiological conditions, activation of a cGMP-dependent pathway moderates the Ca2+i-enhancing action of hypertrophic factors such as angiotensin II. By contrast, a cGMP-independent pathway predominates under pathophysiological conditions when GC-A is desensitized by high ANP levels. The concomitant rise in [Ca2+]i might increase the propensity to cardiac hypertrophy and arrhythmias. PMID:22027011

  4. Cannabinoid inhibition of adenylate cyclase-mediated signal transduction and interleukin 2 (IL-2) expression in the murine T-cell line, EL4.IL-2.

    PubMed

    Condie, R; Herring, A; Koh, W S; Lee, M; Kaminski, N E

    1996-05-31

    Cannabinoid receptors negatively regulate adenylate cyclase through a pertussis toxin-sensitive GTP-binding protein. In the present studies, signaling via the adenylate cyclase/cAMP pathway was investigated in the murine thymoma-derived T-cell line, EL4.IL-2. Northern analysis of EL4.IL-2 cells identified the presence of 4-kilobase CB2 but not CB1 receptor-subtype mRNA transcripts. Southern analysis of genomic DNA digests for the CB2 receptor demonstrated identical banding patterns for EL4.IL-2 cells and mouse-derived DNA, both of which were dissimilar to DNA isolated from rat. Treatment of EL4.IL-2 cells with either cannabinol or Delta9-THC disrupted the adenylate cyclase signaling cascade by inhibiting forskolin-stimulated cAMP accumulation which consequently led to a decrease in protein kinase A activity and the binding of transcription factors to a CRE consensus sequence. Likewise, an inhibition of phorbol 12-myristate 13-acetate (PMA)/ionomycin-induced interleukin 2 (IL-2) protein secretion, which correlated to decreased IL-2 gene transcription, was induced by both cannabinol and Delta9-THC. Further, cannabinoid treatment also decreased PMA/ionomycin-induced nuclear factor binding to the AP-1 proximal site of the IL-2 promoter. Conversely, forskolin enhanced PMA/ionomycin-induced AP-1 binding. These findings suggest that inhibition of signal transduction via the adenylate cyclase/cAMP pathway induces T-cell dysfunction which leads to a diminution in IL-2 gene transcription.

  5. STC1 interference on calcitonin family of receptors signaling during osteoblastogenesis via adenylate cyclase inhibition.

    PubMed

    Terra, Silvia R; Cardoso, João Carlos R; Félix, Rute C; Martins, Leo Anderson M; Souza, Diogo Onofre G; Guma, Fatima C R; Canário, Adelino Vicente M; Schein, Vanessa

    2015-03-05

    Stanniocalcin 1 (STC1) and calcitonin gene-related peptide (CGRP) are involved in bone formation/remodeling. Here we investigate the effects of STC1 on functional heterodimer complex CALCRL/RAMP1, expression and activity during osteoblastogenesis. STC1 did not modify CALCRL and ramp1 gene expression during osteoblastogenesis when compared to controls. However, plasma membrane spatial distribution of CALCRL/RAMP1 was modified in 7-day pre-osteoblasts exposed to either CGRP or STC1, and both peptides induced CALCRL and RAMP1 assembly. CGRP, but not STC1 stimulated cAMP accumulation in 7-day osteoblasts and in CALCRL/RAMP1 transfected HEK293 cells. Furthermore, STC1 inhibited forskolin stimulated cAMP accumulation of HEK293 cells, but not in CALCRL/RAMP1 transfected HEK293 cells. However, STC1 inhibited cAMP accumulation in calcitonin receptor (CTR) HEK293 transfected cells stimulated by calcitonin. In conclusion, STC1 signals through inhibitory G-protein modulates CGRP receptor spatial localization during osteoblastogenesis and may function as a regulatory factor interacting with calcitonin peptide members during bone formation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  6. The Adenylate Cyclase Toxins of Bacillus anthracis and Bordetella pertussis Promote Th2 Cell Development by Shaping T Cell Antigen Receptor Signaling

    PubMed Central

    Rossi Paccani, Silvia; Benagiano, Marisa; Capitani, Nagaja; Zornetta, Irene; Ladant, Daniel; Montecucco, Cesare; D'Elios, Mario M.; Baldari, Cosima T.

    2009-01-01

    The adjuvanticity of bacterial adenylate cyclase toxins has been ascribed to their capacity, largely mediated by cAMP, to modulate APC activation, resulting in the expression of Th2–driving cytokines. On the other hand, cAMP has been demonstrated to induce a Th2 bias when present during T cell priming, suggesting that bacterial cAMP elevating toxins may directly affect the Th1/Th2 balance. Here we have investigated the effects on human CD4+ T cell differentiation of two adenylate cyclase toxins, Bacillus anthracis edema toxin (ET) and Bordetella pertussis CyaA, which differ in structure, mode of cell entry, and subcellular localization. We show that low concentrations of ET and CyaA, but not of their genetically detoxified adenylate cyclase defective counterparts, potently promote Th2 cell differentiation by inducing expression of the master Th2 transcription factors, c-maf and GATA-3. We also present evidence that the Th2–polarizing concentrations of ET and CyaA selectively inhibit TCR–dependent activation of Akt1, which is required for Th1 cell differentiation, while enhancing the activation of two TCR–signaling mediators, Vav1 and p38, implicated in Th2 cell differentiation. This is at variance from the immunosuppressive toxin concentrations, which interfere with the earliest step in TCR signaling, activation of the tyrosine kinase Lck, resulting in impaired CD3ζ phosphorylation and inhibition of TCR coupling to ZAP-70 and Erk activation. These results demonstrate that, notwithstanding their differences in their intracellular localization, which result in focalized cAMP production, both toxins directly affect the Th1/Th2 balance by interfering with the same steps in TCR signaling, and suggest that their adjuvanticity is likely to result from their combined effects on APC and CD4+ T cells. Furthermore, our results strongly support the key role of cAMP in the adjuvanticity of these toxins. PMID:19266022

  7. Controlling fertilization and cAMP signaling in sperm by optogenetics.

    PubMed

    Jansen, Vera; Alvarez, Luis; Balbach, Melanie; Strünker, Timo; Hegemann, Peter; Kaupp, U Benjamin; Wachten, Dagmar

    2015-01-20

    Optogenetics is a powerful technique to control cellular activity by light. The light-gated Channelrhodopsin has been widely used to study and manipulate neuronal activity in vivo, whereas optogenetic control of second messengers in vivo has not been examined in depth. In this study, we present a transgenic mouse model expressing a photoactivated adenylyl cyclase (bPAC) in sperm. In transgenic sperm, bPAC mimics the action of the endogenous soluble adenylyl cyclase (SACY) that is required for motility and fertilization: light-stimulation rapidly elevates cAMP, accelerates the flagellar beat, and, thereby, changes swimming behavior of sperm. Furthermore, bPAC replaces endogenous adenylyl cyclase activity. In mutant sperm lacking the bicarbonate-stimulated SACY activity, bPAC restored motility after light-stimulation and, thereby, enabled sperm to fertilize oocytes in vitro. We show that optogenetic control of cAMP in vivo allows to non-invasively study cAMP signaling, to control behaviors of single cells, and to restore a fundamental biological process such as fertilization.

  8. Biochemical activity and multiple locations of particulate guanylate cyclase in Rhyacophila dorsalis acutidens (Insecta: Trichoptera) provide insights into the cGMP signalling pathway in Malpighian tubules.

    PubMed

    Secca, T; Sciaccaluga, M; Marra, A; Barberini, L; Bicchierai, M C

    2011-04-01

    In insect renal physiology, cGMP and cAMP have important regulatory roles. In Drosophila melanogaster, considered a good model for molecular physiology studies, and in other insects, cGMP and cAMP act as signalling molecules in the Malpighian tubules (MTs). However, many questions related to cyclic nucleotide functions are unsolved in principal cells (PC) and stellate cells (SC), the two cell types that compose the MT. In PC, despite the large body of information available on soluble guanylate cyclase (sGC) in the cGMP pathway, the functional circuit of particulate guanylate cyclase (pGC) remains obscure. In SC, on the other side, the synthesis and physiological role of the cGMP are still unknown. Our biochemical data regarding the presence of cyclic nucleotides in the MTs of Rhyacophila dorsalis acutidens revealed a cGMP level above the 50%, in comparison with the cAMP. The specific activity values for the membrane-bound guanylate cyclase were also recorded, implying that, besides the sGC, pGC is a physiologically relevant source of cGMP in MTs. Cytochemical studies showed ultrastructurally that there was a great deal of pGC on the basolateral membranes of both the principal and stellate cells. In addition, pGC was also detected in the contact zone between the two cell types and in the apical microvillar region of the stellate cells bordering the tubule lumen. The pGC signal is so well represented in PC and, unexpectedly in SC of MTs, that it is possible to hypothesize the existence of still uncharacterized physiological processes regulated by the pGC-cGMP system. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. The Impact of the Nitric Oxide (NO)/Soluble Guanylyl Cyclase (sGC) Signaling Cascade on Kidney Health and Disease: A Preclinical Perspective.

    PubMed

    Krishnan, Shalini M; Kraehling, Jan R; Eitner, Frank; Bénardeau, Agnès; Sandner, Peter

    2018-06-09

    Chronic Kidney Disease (CKD) is a highly prevalent disease with a substantial medical need for new and more efficacious treatments. The Nitric Oxide (NO), soluble guanylyl cyclase (sGC), cyclic guanosine monophosphate (cGMP) signaling cascade regulates various kidney functions. cGMP directly influences renal blood flow, renin secretion, glomerular function, and tubular exchange processes. Downregulation of NO/sGC/cGMP signaling results in severe kidney pathologies such as CKD. Therefore, treatment strategies aiming to maintain or increase cGMP might have beneficial effects for the treatment of progressive kidney diseases. Within this article, we review the NO/sGC/cGMP signaling cascade and its major pharmacological intervention sites. We specifically focus on the currently known effects of cGMP on kidney function parameters. Finally, we summarize the preclinical evidence for kidney protective effects of NO-donors, PDE inhibitors, sGC stimulators, and sGC activators.

  10. Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Pathway Is Induced by Mechanical Load and Reduces the Activity of Hedgehog Signaling in Chondrogenic Micromass Cell Cultures

    PubMed Central

    Juhász, Tamás; Szentléleky, Eszter; Szűcs Somogyi, Csilla; Takács, Roland; Dobrosi, Nóra; Engler, Máté; Tamás, Andrea; Reglődi, Dóra; Zákány, Róza

    2015-01-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurohormone exerting protective function during various stress conditions either in mature or developing tissues. Previously we proved the presence of PACAP signaling elements in chicken limb bud-derived chondrogenic cells in micromass cell cultures. Since no data can be found if PACAP signaling is playing any role during mechanical stress in any tissues, we aimed to investigate its contribution in mechanotransduction during chondrogenesis. Expressions of the mRNAs of PACAP and its major receptor, PAC1 increased, while that of other receptors, VPAC1, VPAC2 decreased upon mechanical stimulus. Mechanical load enhanced the expression of collagen type X, a marker of hypertrophic differentiation of chondrocytes and PACAP addition attenuated this elevation. Moreover, exogenous PACAP also prevented the mechanical load evoked activation of hedgehog signaling: protein levels of Sonic and Indian Hedgehogs and Gli1 transcription factor were lowered while expressions of Gli2 and Gli3 were elevated by PACAP application during mechanical load. Our results suggest that mechanical load activates PACAP signaling and exogenous PACAP acts against the hypertrophy inducing effect of mechanical load. PMID:26230691

  11. Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Pathway Is Induced by Mechanical Load and Reduces the Activity of Hedgehog Signaling in Chondrogenic Micromass Cell Cultures.

    PubMed

    Juhász, Tamás; Szentléleky, Eszter; Somogyi, Csilla Szűcs; Takács, Roland; Dobrosi, Nóra; Engler, Máté; Tamás, Andrea; Reglődi, Dóra; Zákány, Róza

    2015-07-29

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurohormone exerting protective function during various stress conditions either in mature or developing tissues. Previously we proved the presence of PACAP signaling elements in chicken limb bud-derived chondrogenic cells in micromass cell cultures. Since no data can be found if PACAP signaling is playing any role during mechanical stress in any tissues, we aimed to investigate its contribution in mechanotransduction during chondrogenesis. Expressions of the mRNAs of PACAP and its major receptor, PAC1 increased, while that of other receptors, VPAC1, VPAC2 decreased upon mechanical stimulus. Mechanical load enhanced the expression of collagen type X, a marker of hypertrophic differentiation of chondrocytes and PACAP addition attenuated this elevation. Moreover, exogenous PACAP also prevented the mechanical load evoked activation of hedgehog signaling: protein levels of Sonic and Indian Hedgehogs and Gli1 transcription factor were lowered while expressions of Gli2 and Gli3 were elevated by PACAP application during mechanical load. Our results suggest that mechanical load activates PACAP signaling and exogenous PACAP acts against the hypertrophy inducing effect of mechanical load.

  12. Pituitary adenylate cyclase-activating polypeptide (PACAP) signaling in the prefrontal cortex modulates cued fear learning, but not spatial working memory, in female rats.

    PubMed

    Kirry, Adam J; Herbst, Matthew R; Poirier, Sarah E; Maskeri, Michelle M; Rothwell, Amy C; Twining, Robert C; Gilmartin, Marieke R

    2018-05-01

    A genetic polymorphism within the gene encoding the pituitary adenylate cyclase- activating polypeptide (PACAP) receptor type I (PAC1R) has recently been associated with hyper-reactivity to threat-related cues in women, but not men, with post-traumatic stress disorder (PTSD). PACAP is a highly conserved peptide, whose role in mediating adaptive physiological stress responses is well established. Far less is understood about the contribution of PACAP signaling in emotional learning and memory, particularly the encoding of fear to discrete cues. Moreover, a neurobiological substrate that may account for the observed link between PAC1R and PTSD in women, but not men, has yet to be identified. Sex differences in PACAP signaling during emotional learning could provide novel targets for the treatment of PTSD. Here we investigated the contribution of PAC1R signaling within the prefrontal cortex to the acquisition of cued fear in female and male rats. We used a variant of fear conditioning called trace fear conditioning, which requires sustained attention to fear cues and depends on working-memory like neuronal activity within the prefrontal cortex. We found that cued fear learning, but not spatial working memory, was impaired by administration of a PAC1R antagonist directly into the prelimbic area of the prefrontal cortex. This effect was specific to females. We also found that levels of mRNA for the PAC1R receptor in the prelimbic cortex were greater in females compared with males, and were highest during and immediately following the proestrus stage of the estrous cycle. Together, these results demonstrate a sex-specific role of PAC1R signaling in learning about threat-related cues. Copyright © 2018 Elsevier Ltd. All rights reserved.

  13. Motile cilia of human airway epithelia contain hedgehog signaling components that mediate noncanonical hedgehog signaling.

    PubMed

    Mao, Suifang; Shah, Alok S; Moninger, Thomas O; Ostedgaard, Lynda S; Lu, Lin; Tang, Xiao Xiao; Thornell, Ian M; Reznikov, Leah R; Ernst, Sarah E; Karp, Philip H; Tan, Ping; Keshavjee, Shaf; Abou Alaiwa, Mahmoud H; Welsh, Michael J

    2018-02-06

    Differentiated airway epithelia produce sonic hedgehog (SHH), which is found in the thin layer of liquid covering the airway surface. Although previous studies showed that vertebrate HH signaling requires primary cilia, as airway epithelia mature, the cells lose primary cilia and produce hundreds of motile cilia. Thus, whether airway epithelia have apical receptors for SHH has remained unknown. We discovered that motile cilia on airway epithelial cells have HH signaling proteins, including patched and smoothened. These cilia also have proteins affecting cAMP-dependent signaling, including Gα i and adenylyl cyclase 5/6. Apical SHH decreases intracellular levels of cAMP, which reduces ciliary beat frequency and pH in airway surface liquid. These results suggest that apical SHH may mediate noncanonical HH signaling through motile cilia to dampen respiratory defenses at the contact point between the environment and the lung, perhaps counterbalancing processes that stimulate airway defenses. Copyright © 2018 the Author(s). Published by PNAS.

  14. Pituitary adenylate cyclase activating polypeptide reduces A-type K+ currents and caspase activity in cultured adult mouse olfactory neurons.

    PubMed

    Han, P; Lucero, M T

    2005-01-01

    Pituitary adenylate cyclase activating polypeptide has been shown to reduce apoptosis in neonatal cerebellar and olfactory receptor neurons, however the underlying mechanisms have not been elucidated. In addition, the neuroprotective effects of pituitary adenylate cyclase activating polypeptide have not been examined in adult tissues. To study the effects of pituitary adenylate cyclase activating polypeptide on neurons in apoptosis, we measured caspase activation in adult olfactory receptor neurons in vitro. Interestingly, we found that the protective effects of pituitary adenylate cyclase activating polypeptide were related to the absence of a 4-aminopyridine (IC50=144 microM) sensitive rapidly inactivating potassium current often referred to as A-type current. In the presence of 40 nM pituitary adenylate cyclase activating polypeptide 38, both A-type current and activated caspases were significantly reduced. A-type current reduction by pituitary adenylate cyclase activating polypeptide was blocked by inhibiting the phospholipase C pathway, but not the adenylyl cyclase pathway. Our observation that 5 mM 4-aminopyridine mimicked the caspase inhibiting effects of pituitary adenylate cyclase activating polypeptide indicates that A-type current is involved in apoptosis. This work contributes to our growing understanding that potassium currents are involved with the activation of caspases to affect the balance between cell life and death.

  15. Toll-like Receptor 4 Signaling Confers Cardiac Protection Against Ischemic Injury via Inducible Nitric Oxide Synthase- and Soluble Guanylate Cyclase-dependent Mechanisms

    PubMed Central

    Wang, E; Feng, Yan; Zhang, Ming; Zou, Lin; Li, Yan; Buys, Emmanuel S.; Huang, Peigen; Brouckaert, Peter; Chao, Wei

    2011-01-01

    Background Prior administration of a small dose of lipopolysaccharide confers a cardiac protection against ischemia-reperfusion injury. However, the signaling mechanisms that control the protection are incompletely understood. We tested the hypothesis that TLR4 mediates the ability of lipopolysaccharide to protect against cardiac ischemia-reperfusion injury through distinct intracellular pathways involving myeloid differentiation factor 88 (MyD88), TIR-domain-containing adaptor protein inducing interferon-β–mediated transcription-factor (Trif), inducible nitric-oxide synthase (iNOS), and soluble guanylate cyclase (sGC). Methods Wild-type mice and the genetically modified mice, i.e., TLR4-deficient (TLR4-def), TLR2 knockout (TLR2−/−), MyD88−/−, Trif−/−, iNOS−/−, and sGCα1−/−, were treated with normal saline or 0.1 mg/kg of lipopolysaccharide, intraperitoneally. Twenty-four hours later, isolated hearts were perfused in a Langendorff apparatus and subsequently subjected to 30 min of global ischemia and reperfusion for up to 60 min. Left ventricular function and myocardial infarction sizes were examined. Results Compared to saline-treated mice, lipopolysaccharide-treated mice had markedly improved left ventricular developed pressure and dP/dtmax (P < 0.01) and reduced MI sizes (37.2 ± 3.4% vs. 19.8 ± 4.9%, P < 0.01) after ischemia-reperfusion. The cardiac protective effect of lipopolysaccharide was abolished in the TLR4-def and MyD88−/− mice, but remained intact in TLR2−/− or Trif−/− mice. iNOS−/− mice or wild-type mice treated with the iNOS inhibitor 1400W failed to respond to the TLR4-induced nitric oxide production and were not protected by the lipopolysaccharide preconditioning. While sGC 1−/− mice had robust nitric oxide production in response to lipopolysaccharide, they were not protected by the TLR4-elicited cardiac protection. Conclusions TLR4 activation confers a potent cardiac protection against ischemia

  16. ADP-ribosyl cyclases regulate early development of the sea urchin.

    PubMed

    Ramakrishnan, Latha; Uhlinger, Kevin; Dale, Leslie; Hamdoun, Amro; Patel, Sandip

    2016-06-01

    ADP-ribosyl cyclases are multifunctional enzymes involved in the metabolism of nucleotide derivatives necessary for Ca 2+ signalling such as cADPR and NAADP. Although Ca 2+ signalling is a critical regulator of early development, little is known of the role of ADP-ribosyl cyclases during embryogenesis. Here we analyze the expression, activity and function of ADP-ribosyl cyclases in the embryo of the sea urchin - a key organism for study of both Ca 2+ signalling and embryonic development. ADP-ribosyl cyclase isoforms (SpARC1-4) showed unique changes in expression during early development. These changes were associated with an increase in the ratio of cADPR:NAADP production. Over-expression of SpARC4 (a preferential cyclase) disrupted gastrulation. Our data highlight the importance of ADP-ribosyl cyclases during embryogenesis.

  17. Stimulation of Hippocampal Adenylyl Cyclase Activity Dissociates Memory Consolidation Processes for Response and Place Learning

    ERIC Educational Resources Information Center

    Martel, Guillaume; Millard, Annabelle; Jaffard, Robert; Guillou, Jean-Louis

    2006-01-01

    Procedural and declarative memory systems are postulated to interact in either a synergistic or a competitive manner, and memory consolidation appears to be a highly critical stage for this process. However, the precise cellular mechanisms subserving these interactions remain unknown. To investigate this issue, 24-h retention performances were…

  18. A Novel Ras-interacting Protein Required for Chemotaxis and Cyclic Adenosine Monophosphate Signal Relay in Dictyostelium

    PubMed Central

    Lee, Susan; Parent, Carole A.; Insall, Robert; Firtel, Richard A.

    1999-01-01

    We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced ∼60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras-regulated pathway

  19. A novel Ras-interacting protein required for chemotaxis and cyclic adenosine monophosphate signal relay in Dictyostelium.

    PubMed

    Lee, S; Parent, C A; Insall, R; Firtel, R A

    1999-09-01

    We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced approximately 60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras

  20. Pituitary adenylate cyclase-activating polypeptide stimulates glucose production via the hepatic sympathetic innervation in rats.

    PubMed

    Yi, Chun-Xia; Sun, Ning; Ackermans, Mariette T; Alkemade, Anneke; Foppen, Ewout; Shi, Jing; Serlie, Mireille J; Buijs, Ruud M; Fliers, Eric; Kalsbeek, Andries

    2010-07-01

    The unraveling of the elaborate brain networks that control glucose metabolism presents one of the current challenges in diabetes research. Within the central nervous system, the hypothalamus is regarded as the key brain area to regulate energy homeostasis. The aim of the present study was to investigate the hypothalamic mechanism involved in the hyperglycemic effects of the neuropeptide pituitary adenylyl cyclase-activating polypeptide (PACAP). Endogenous glucose production (EGP) was determined during intracerebroventricular infusions of PACAP-38, vasoactive intestinal peptide (VIP), or their receptor agonists. The specificity of their receptors was examined by coinfusions of receptor antagonists. The possible neuronal pathway involved was investigated by 1) local injections in hypothalamic nuclei, 2) retrograde neuronal tracing from the thoracic spinal cord to hypothalamic preautonomic neurons together with Fos immunoreactivity, and 3) specific hepatic sympathetic or parasympathetic denervation to block the autonomic neuronal input to liver. Intracerebroventricular infusion of PACAP-38 increased EGP to a similar extent as a VIP/PACAP-2 (VPAC2) receptor agonist, and intracerebroventricular administration of VIP had significantly less influence on EGP. The PACAP-38 induced increase of EGP was significantly suppressed by preinfusion of a VPAC2 but not a PAC1 receptor antagonist, as well as by hepatic sympathetic but not parasympathetic denervation. In the hypothalamus, Fos immunoreactivity induced by PACAP-38 was colocalized within autonomic neurons in paraventricular nuclei projecting to preganglionic sympathetic neurons in the spinal cord. Local infusion of PACAP-38 directly into the PVN induced a significant increase of EGP. This study demonstrates that PACAP-38 signaling via sympathetic preautonomic neurons located in the paraventricular nucleus is an important component in the hypothalamic control of hepatic glucose production.

  1. An Odor-Specific Threshold Deficit Implicates Abnormal Intracellular Cyclic AMP Signaling in Schizophrenia

    PubMed Central

    Turetsky, Bruce I.; Moberg, Paul J.

    2012-01-01

    Objective Although olfactory deficits are common in schizophrenia, their underlying pathophysiology remains unknown. Recent evidence has suggested that cAMP signaling may be disrupted in schizophrenia. Since cAMP mediates signal transduction in olfactory receptor neurons, this could contribute to the etiology of observed olfactory deficits. This study was designed to test this hypothesis by determining odor detection threshold sensitivities to two odorants that differ in their relative activations of this intracellular cAMP signaling cascade. Method Thirty schizophrenia patients, 25 healthy comparison subjects, and 19 unaffected first-degree relatives of schizophrenia patients were studied. Odor detection threshold sensitivities were measured for the two odorants citralva and lyral. Although both have fruity/floral scents, citralva strongly activates adenylyl cyclase to increase cAMP levels, while lyral is a very weak activator of adenylyl cyclase. Results There was a significant group-by-odor interaction. Both schizophrenia patients and unaffected first-degree relatives were impaired in their ability to detect lyral versus citralva. Comparison subjects were equally sensitive to both odorants. This selective deficit could not be explained by differences in age, sex, smoking, clinical symptom profile, or medication use. Conclusions This study establishes the presence of an odor-specific hyposmia that may denote a disruption of cAMP-mediated signal transduction in schizophrenia. The presence of a parallel deficit in the patients’ unaffected first-degree relatives suggests that this deficit is genetically mediated. Although additional physiological studies are needed to confirm the underlying mechanism, these results offer strong inferential support for the hypothesis that cAMP signaling is dys-regulated in schizophrenia. PMID:19074977

  2. An odor-specific threshold deficit implicates abnormal intracellular cyclic AMP signaling in schizophrenia.

    PubMed

    Turetsky, Bruce I; Moberg, Paul J

    2009-02-01

    Although olfactory deficits are common in schizophrenia, their underlying pathophysiology remains unknown. Recent evidence has suggested that cAMP signaling may be disrupted in schizophrenia. Since cAMP mediates signal transduction in olfactory receptor neurons, this could contribute to the etiology of observed olfactory deficits. This study was designed to test this hypothesis by determining odor detection threshold sensitivities to two odorants that differ in their relative activations of this intracellular cAMP signaling cascade. Thirty schizophrenia patients, 25 healthy comparison subjects, and 19 unaffected first-degree relatives of schizophrenia patients were studied. Odor detection threshold sensitivities were measured for the two odorants citralva and lyral. Although both have fruity/floral scents, citralva strongly activates adenylyl cyclase to increase cAMP levels, while lyral is a very weak activator of adenylyl cyclase. There was a significant group-by-odor interaction. Both schizophrenia patients and unaffected first-degree relatives were impaired in their ability to detect lyral versus citralva. Comparison subjects were equally sensitive to both odorants. This selective deficit could not be explained by differences in age, sex, smoking, clinical symptom profile, or medication use. This study establishes the presence of an odor-specific hyposmia that may denote a disruption of cAMP-mediated signal transduction in schizophrenia. The presence of a parallel deficit in the patients' unaffected first-degree relatives suggests that this deficit is genetically mediated. Although additional physiological studies are needed to confirm the underlying mechanism, these results offer strong inferential support for the hypothesis that cAMP signaling is dysregulated in schizophrenia.

  3. Evolution and cell physiology. 2. The evolution of cell signaling: from mitochondria to Metazoa.

    PubMed

    Blackstone, Neil W

    2013-11-01

    The history of life is a history of levels-of-selection transitions. Each transition requires mechanisms that mediate conflict among the lower-level units. In the origins of multicellular eukaryotes, cell signaling is one such mechanism. The roots of cell signaling, however, may extend to the previous major transition, the origin of eukaryotes. Energy-converting protomitochondria within a larger cell allowed eukaryotes to transcend the surface-to-volume constraints inherent in the design of prokaryotes. At the same time, however, protomitochondria can selfishly allocate energy to their own replication. Metabolic signaling may have mediated this principal conflict in several ways. Variation of the protomitochondria was constrained by stoichiometry and strong metabolic demand (state 3) exerted by the protoeukaryote. Variation among protoeukaryotes was increased by the sexual stage of the life cycle, triggered by weak metabolic demand (state 4), resulting in stochastic allocation of protomitochondria to daughter cells. Coupled with selection, many selfish protomitochondria could thus be removed from the population. Hence, regulation of states 3 and 4, as, for instance, provided by the CO2/soluble adenylyl cyclase/cAMP pathway in mitochondria, was critical for conflict mediation. Subsequently, as multicellular eukaryotes evolved, metabolic signaling pathways employed by eukaryotes to mediate conflict within cells could now be co-opted into conflict mediation between cells. For example, in some fungi, the CO2/soluble adenylyl cyclase/cAMP pathway regulates the transition from yeast to forms with hyphae. In animals, this pathway regulates the maturation of sperm. While the particular features (sperm and hyphae) are distinct, both may involve between-cell conflicts that required mediation.

  4. Adenylylation of Tyr77 stabilizes Rab1b GTPase in an active state: A molecular dynamics simulation analysis

    PubMed Central

    Luitz, Manuel P.; Bomblies, Rainer; Ramcke, Evelyn; Itzen, Aymelt; Zacharias, Martin

    2016-01-01

    The pathogenic pathway of Legionella pneumophila exploits the intercellular vesicle transport system via the posttranslational attachment of adenosine monophosphate (AMP) to the Tyr77 sidechain of human Ras like GTPase Rab1b. The modification, termed adenylylation, is performed by the bacterial enzyme DrrA/SidM, however the effect on conformational properties of the molecular switch mechanism of Rab1b remained unresolved. In this study we find that the adenylylation of Tyr77 stabilizes the active Rab1b state by locking the switch in the active signaling conformation independent of bound GTP or GDP and that electrostatic interactions due to the additional negative charge in the switch region make significant contributions. The stacking interaction between adenine and Phe45 however, seems to have only minor influence on this stabilisation. The results may also have implications for the mechanistic understanding of conformational switching in other signaling proteins. PMID:26818796

  5. Regulation of Sertoli cell tight junction dynamics in the rat testis via the nitric oxide synthase/soluble guanylate cyclase/3',5'-cyclic guanosine monophosphate/protein kinase G signaling pathway: an in vitro study.

    PubMed

    Lee, Nikki P Y; Cheng, C Yan

    2003-07-01

    barrier assembly. Cytokines, such as TGF-beta and TNF-alpha, known to perturb the Sertoli cell TJ barrier, were also shown to stimulate Sertoli cell iNOS and eNOS expression dose dependently in vitro. Collectively, these results illustrate NOS is an important physiological regulator of TJ dynamics in the testis, exerting its effects via the NO/soluble guanylate cyclase/cGMP/protein kinase G signaling pathway.

  6. Signaling of pigment-dispersing factor (PDF) in the Madeira cockroach Rhyparobia maderae.

    PubMed

    Wei, Hongying; Yasar, Hanzey; Funk, Nico W; Giese, Maria; Baz, El-Sayed; Stengl, Monika

    2014-01-01

    The insect neuropeptide pigment-dispersing factor (PDF) is a functional ortholog of vasoactive intestinal polypeptide, the coupling factor of the mammalian circadian pacemaker. Despite of PDF's importance for synchronized circadian locomotor activity rhythms its signaling is not well understood. We studied PDF signaling in primary cell cultures of the accessory medulla, the circadian pacemaker of the Madeira cockroach. In Ca²⁺ imaging studies four types of PDF-responses were distinguished. In regularly bursting type 1 pacemakers PDF application resulted in dose-dependent long-lasting increases in Ca²⁺ baseline concentration and frequency of oscillating Ca²⁺ transients. Adenylyl cyclase antagonists prevented PDF-responses in type 1 cells, indicating that PDF signaled via elevation of intracellular cAMP levels. In contrast, in type 2 pacemakers PDF transiently raised intracellular Ca²⁺ levels even after blocking adenylyl cyclase activity. In patch clamp experiments the previously characterized types 1-4 could not be identified. Instead, PDF-responses were categorized according to ion channels affected. Application of PDF inhibited outward potassium or inward sodium currents, sometimes in the same neuron. In a comparison of Ca²⁺ imaging and patch clamp experiments we hypothesized that in type 1 cells PDF-dependent rises in cAMP concentrations block primarily outward K⁺ currents. Possibly, this PDF-dependent depolarization underlies PDF-dependent phase advances of pacemakers. Finally, we propose that PDF-dependent concomitant modulation of K⁺ and Na⁺ channels in coupled pacemakers causes ultradian membrane potential oscillations as prerequisite to efficient synchronization via resonance.

  7. Signaling of Pigment-Dispersing Factor (PDF) in the Madeira Cockroach Rhyparobia maderae

    PubMed Central

    Funk, Nico W.; Giese, Maria; Baz, El-Sayed; Stengl, Monika

    2014-01-01

    The insect neuropeptide pigment-dispersing factor (PDF) is a functional ortholog of vasoactive intestinal polypeptide, the coupling factor of the mammalian circadian pacemaker. Despite of PDF's importance for synchronized circadian locomotor activity rhythms its signaling is not well understood. We studied PDF signaling in primary cell cultures of the accessory medulla, the circadian pacemaker of the Madeira cockroach. In Ca2+ imaging studies four types of PDF-responses were distinguished. In regularly bursting type 1 pacemakers PDF application resulted in dose-dependent long-lasting increases in Ca2+ baseline concentration and frequency of oscillating Ca2+ transients. Adenylyl cyclase antagonists prevented PDF-responses in type 1 cells, indicating that PDF signaled via elevation of intracellular cAMP levels. In contrast, in type 2 pacemakers PDF transiently raised intracellular Ca2+ levels even after blocking adenylyl cyclase activity. In patch clamp experiments the previously characterized types 1–4 could not be identified. Instead, PDF-responses were categorized according to ion channels affected. Application of PDF inhibited outward potassium or inward sodium currents, sometimes in the same neuron. In a comparison of Ca2+ imaging and patch clamp experiments we hypothesized that in type 1 cells PDF-dependent rises in cAMP concentrations block primarily outward K+ currents. Possibly, this PDF-dependent depolarization underlies PDF-dependent phase advances of pacemakers. Finally, we propose that PDF-dependent concomitant modulation of K+ and Na+ channels in coupled pacemakers causes ultradian membrane potential oscillations as prerequisite to efficient synchronization via resonance. PMID:25269074

  8. DISPARATE DEVELOPMENTAL NEUROTOXICANTS CONVERGE ON THE CYCLIC AMP SIGNALING CASCADE, REVEALED BY TRANSCRIPTIONAL PROFILES IN VITRO AND IN VIVO

    PubMed Central

    Adigun, Abayomi A.; Seidler, Frederic J.; Slotkin, Theodore A.

    2009-01-01

    Cell-signaling cascades are convergent targets for developmental neurotoxicity of otherwise unrelated agents. We compared organophosphates (chlorpyrifos, diazinon), an organochlorine (dieldrin) and a metal (Ni2+) for their effects on neuronotypic PC12 cells, assessing gene transcription involved in the cyclic AMP pathway. Each agent was introduced during neurodifferentiation at a concentration of 30 μM for 24 or 72 hr and we assessed 69 genes encoding adenylyl cyclase isoforms and regulators, G-protein α- and β,γ-subunits, protein kinase A subtypes and the phosphodiesterase family. We found strong concordance among the four agents across all the gene families, with the strongest relationships for the G-proteins, followed by adenylyl cyclase, and lesser concordance for protein kinase A and phosphodiesterase. Superimposed on this pattern, chlorpyrifos and diazinon were surprisingly the least alike, whereas there was strong concordance of dieldrin and Ni2+ with each other and with each individual organophosphate. Further, the effects of chlorpyrifos differed substantially depending on whether cells were undifferentiated or differentiating. To resolve the disparities between chlorpyrifos and diazinon, we performed analyses in rat brain regions after in vivo neonatal exposures; unlike the in vitro results, there was strong concordance. Our results show that unrelated developmental neurotoxicants can nevertheless produce similar outcomes by targeting cell signaling pathways involved in neurodifferentiation during a critical developmental period of vulnerability. Nevertheless, a full evaluation of concordance between different toxicants requires evaluations of in vitro systems that detect direct effects, as well as in vivo systems that allow for more complex interactions that converge on the same pathway. PMID:20026089

  9. The guanylyl cyclase family at Y2K.

    PubMed

    Wedel, B; Garbers, D

    2001-01-01

    During the 1980s the purification, cloning, and expression of various forms of guanylyl cyclase (GC) revealed that they served as receptors for extracellular signals. Seven membrane forms, which presumably exist as homodimers, and four subunits of apparent heterodimers (commonly referred to as the soluble forms) are known, but in animals such as nematodes, much larger numbers of GCs are expressed. The number of transmembrane segments (none, one, or multiple) divide the GC family into three groups. Those with no or one transmembrane segment bind nitric oxide/carbon monoxide (NO/CO) or peptides. There are no known ligands for the multiple transmembrane segment class of GCs. Mutational and structural analyses support a model where catalysis requires a shared substrate binding site between the subunits, whether homomeric or heteromeric in nature. Because some cyclases or cyclase ligand genes lack specific GC inhibitors, disruption of either has been used to define the functions of individual cyclases, as well as to define human genetic disease counterparts.

  10. Inhibition of Gαs/cAMP Signaling Decreases TCR-Stimulated IL-2 transcription in CD4(+) T Helper Cells.

    PubMed

    Hynes, Thomas R; Yost, Evan A; Yost, Stacy M; Hartle, Cassandra M; Ott, Braden J; Berlot, Catherine H

    2015-07-06

    The role of cAMP in regulating T cell activation and function has been controversial. cAMP is generally known as an immunosuppressant, but it is also required for generating optimal immune responses. As the effect of cAMP is likely to depend on its cellular context, the current study investigated whether the mechanism of activation of Gαs and adenylyl cyclase influences their effect on T cell receptor (TCR)-stimulated interleukin-2 (IL-2) mRNA levels. The effect of blocking Gs-coupled receptor (GsPCR)-mediated Gs activation on TCR-stimulated IL-2 mRNA levels in CD4(+) T cells was compared with that of knocking down Gαs expression or inhibiting adenylyl cyclase activity. The effect of knocking down Gαs expression on TCR-stimulated cAMP accumulation was compared with that of blocking GsPCR signaling. ZM-241385, an antagonist to the Gs-coupled A2A adenosine receptor (A2AR), enhanced TCR-stimulated IL-2 mRNA levels in primary human CD4(+) T helper cells and in Jurkat T cells. A dominant negative Gαs construct, GαsDN3, also enhanced TCR-stimulated IL-2 mRNA levels. Similar to GsPCR antagonists, GαsDN3 blocked GsPCR-dependent activation of both Gαs and Gβγ. In contrast, Gαs siRNA and 2',5'-dideoxyadenosine (ddA), an adenylyl cyclase inhibitor, decreased TCR-stimulated IL-2 mRNA levels. Gαs siRNA, but not GαsDN3, decreased TCR-stimulated cAMP synthesis. Potentiation of IL-2 mRNA levels by ZM-241385 required at least two days of TCR stimulation, and addition of ddA after three days of TCR stimulation enhanced IL-2 mRNA levels. GsPCRs play an inhibitory role in the regulation of TCR-stimulated IL-2 mRNA levels whereas Gαs and cAMP can play a stimulatory one. Additionally, TCR-dependent activation of Gαs does not appear to involve GsPCRs. These results suggest that the context of Gαs/cAMP activation and the stage of T cell activation and differentiation determine the effect on TCR-stimulated IL-2 mRNA levels.

  11. Time course of the uridylylation and adenylylation states in the glutamine synthetase bicyclic cascade.

    PubMed Central

    Varón-Castellanos, R; Havsteen, B H; García-Moreno, M; Valero-Ruiz, E; Molina-Alarcón, M; García-Cánovas, F

    1993-01-01

    A kinetic analysis of the glutamine synthetase bicyclic cascade is presented. It includes the dependence on time from the onset of the reaction of both the uridylylation of Shapiro's regulatory protein and the adenylylation of the glutamine synthetase. The transient phase equations obtained allow an estimation of the time elapsed until the states of uridylylation and adenylylation reach their steady-states, and therefore an evaluation of the effective sensitivity of the system. The contribution of the uridylylation cycle to the adenylylation cycle has been studied, and an equation relating the state of adenylylation at any time to the state of uridylylation at the same instant has been derived. PMID:8104399

  12. The Dictyostelium MAP kinase kinase DdMEK1 regulates chemotaxis and is essential for chemoattractant-mediated activation of guanylyl cyclase.

    PubMed Central

    Ma, H; Gamper, M; Parent, C; Firtel, R A

    1997-01-01

    We have identified a MAP kinase kinase (DdMEK1) that is required for proper aggregation in Dictyostelium. Null mutations produce extremely small aggregate sizes, resulting in the formation of slugs and terminal fruiting bodies that are significantly smaller than those of wild-type cells. Time-lapse video microscopy and in vitro assays indicate that the cells are able to produce cAMP waves that move through the aggregation domains. However, these cells are unable to undergo chemotaxis properly during aggregation in response to the chemoattractant cAMP or activate guanylyl cyclase, a known regulator of chemotaxis in Dictyostelium. The activation of guanylyl cyclase in response to osmotic stress is, however, normal. Expression of putative constitutively active forms of DdMEK1 in a ddmek1 null background is capable, at least partially, of complementing the small aggregate size defect and the ability to activate guanylyl cyclase. However, this does not result in constitutive activation of guanylyl cyclase, suggesting that DdMEK1 activity is necessary, but not sufficient, for cAMP activation of guanylyl cyclase. Analysis of a temperature-sensitive DdMEK1 mutant suggests that DdMEK1 activity is required throughout aggregation at the time of guanylyl cyclase activation, but is not essential for proper morphogenesis during the later multicellular stages. The activation of the MAP kinase ERK2, which is essential for chemoattractant activation of adenylyl cyclase, is not affected in ddmek1 null strains, indicating that DdMEK1 does not regulate ERK2 and suggesting that at least two independent MAP kinase cascades control aggregation in Dictyostelium. PMID:9250676

  13. New functional activity of aripiprazole revealed: robust antagonism of D2 dopamine receptor-stimulated Gβγ signaling

    PubMed Central

    Brust, Tarsis F.; Hayes, Michael P.; Roman, David L.; Watts, Val J.

    2014-01-01

    The dopamine D2 receptor (DRD2) is a G protein-coupled receptor (GPCR) that is generally considered to be a primary target in the treatment of schizophrenia. First generation antipsychotic drugs (e.g. haloperidol) are antagonists of the DRD2, while second generation antipsychotic drugs (e.g. olanzapine) antagonize DRD2 and 5HT2A receptors. Notably, both these classes of drugs may cause side effects associated with D2 receptor antagonism (e.g. hyperprolactemia and extrapyramidal symptoms). The novel, “third generation” antipsychotic drug, aripiprazole is also used to treat schizophrenia, with the remarkable advantage that its tendency to cause extrapyramidal symptoms is minimal. Aripiprazole is considered a partial agonist of the DRD2, but it also has partial agonist/antagonist activity for other GPCRs. Further, aripiprazole has been reported to have a unique activity profile in functional assays with the DRD2. In the present study the molecular pharmacology of aripiprazole was further examined in HEK cell models stably expressing the DRD2 and specific isoforms of adenylyl cyclase to assess functional responses of Gα and Gβγ subunits. Additional studies examined the activity of aripiprazole in DRD2-mediated heterologous sensitization of adenylyl cyclase and cell-based dynamic mass redistribution (DMR). Aripiprazole displayed a unique functional profile for modulation of G proteins, being a partial agonist for Gαi/o and a robust antagonist for Gβγ signaling. Additionally, aripiprazole was a weak partial agonist for both heterologous sensitization and dynamic mass redistribution. PMID:25449598

  14. Identification of a Novel Gnao-Mediated Alternate Olfactory Signaling Pathway in Murine OSNs.

    PubMed

    Scholz, Paul; Mohrhardt, Julia; Jansen, Fabian; Kalbe, Benjamin; Haering, Claudia; Klasen, Katharina; Hatt, Hanns; Osterloh, Sabrina

    2016-01-01

    It is generally agreed that in olfactory sensory neurons (OSNs), the binding of odorant molecules to their specific olfactory receptor (OR) triggers a cAMP-dependent signaling cascade, activating cyclic-nucleotide gated (CNG) channels. However, considerable controversy dating back more than 20 years has surrounded the question of whether alternate signaling plays a role in mammalian olfactory transduction. In this study, we demonstrate a specific alternate signaling pathway in Olfr73-expressing OSNs. Methylisoeugenol (MIEG) and at least one other known weak Olfr73 agonist (Raspberry Ketone) trigger a signaling cascade independent from the canonical pathway, leading to the depolarization of the cell. Interestingly, this pathway is mediated by Gnao activation, leading to Cl(-) efflux; however, the activation of adenylyl cyclase III (ACIII), the recruitment of Ca(2+) from extra-or intracellular stores, and phosphatidylinositol 3-kinase-dependent signaling (PI signaling) are not involved. Furthermore, we demonstrated that our newly identified pathway coexists with the canonical olfactory cAMP pathway in the same OSN and can be triggered by the same OR in a ligand-selective manner. We suggest that this pathway might reflect a mechanism for odor recognition predominantly used in early developmental stages before olfactory cAMP signaling is fully developed. Taken together, our findings support the existence of at least one odor-induced alternate signal transduction pathway in native OSNs mediated by Olfr73 in a ligand-selective manner.

  15. Spatiotemporal control of opioid signaling and behavior.

    PubMed

    Siuda, Edward R; Copits, Bryan A; Schmidt, Martin J; Baird, Madison A; Al-Hasani, Ream; Planer, William J; Funderburk, Samuel C; McCall, Jordan G; Gereau, Robert W; Bruchas, Michael R

    2015-05-20

    Optogenetics is now a widely accepted tool for spatiotemporal manipulation of neuronal activity. However, a majority of optogenetic approaches use binary on/off control schemes. Here, we extend the optogenetic toolset by developing a neuromodulatory approach using a rationale-based design to generate a Gi-coupled, optically sensitive, mu-opioid-like receptor, which we term opto-MOR. We demonstrate that opto-MOR engages canonical mu-opioid signaling through inhibition of adenylyl cyclase, activation of MAPK and G protein-gated inward rectifying potassium (GIRK) channels and internalizes with kinetics similar to that of the mu-opioid receptor. To assess in vivo utility, we expressed a Cre-dependent viral opto-MOR in RMTg/VTA GABAergic neurons, which led to a real-time place preference. In contrast, expression of opto-MOR in GABAergic neurons of the ventral pallidum hedonic cold spot led to real-time place aversion. This tool has generalizable application for spatiotemporal control of opioid signaling and, furthermore, can be used broadly for mimicking endogenous neuronal inhibition pathways. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Spatiotemporal control of opioid signaling and behavior

    PubMed Central

    Siuda, Edward R.; Copits, Bryan A.; Schmidt, Martin J.; Baird, Madison A.; Al-Hasani, Ream; Planer, William J.; Funderburk, Samuel C.; McCall, Jordan G.; Gereau, Robert W.; Bruchas, Michael R.

    2015-01-01

    Summary Optogenetics is now a widely accepted tool for spatiotemporal manipulation of neuronal activity. However, a majority of optogenetic approaches use binary on/off control schemes. Here we extend the optogenetic toolset by developing a neuromodulatory approach using a rationale-based design to generate a Gi-coupled, optically-sensitive, mu-opioid-like receptor, we term opto-MOR. We demonstrate that opto-MOR engages canonical mu-opioid signaling through inhibition of adenylyl cyclase, activation of MAPK and G protein-gated inward rectifying potassium (GIRK) channels, and internalizes with similar kinetics as the mu-opioid receptor. To assess in vivo utility we expressed a Cre-dependent viral opto-MOR in RMTg/VTA GABAergic neurons, which led to a real-time place preference. In contrast, expression of opto-MOR in GABAergic neurons of the ventral pallidum hedonic cold spot, led to real-time place aversion. This tool has generalizable application for spatiotemporal control of opioid signaling and, furthermore, can be used broadly for mimicking endogenous neuronal inhibition pathways. PMID:25937173

  17. Recruitment of endosomal signaling mediates the forskolin modulation of guinea pig cardiac neuron excitability.

    PubMed

    Hardwick, Jean C; Clason, Todd A; Tompkins, John D; Girard, Beatrice M; Baran, Caitlin N; Merriam, Laura A; May, Victor; Parsons, Rodney L

    2017-08-01

    Forskolin, a selective activator of adenylyl cyclase (AC), commonly is used to establish actions of G protein-coupled receptors (GPCRs) that are initiated primarily through activation of AC/cAMP signaling pathways. In the present study, forskolin was used to evaluate the potential role of AC/cAMP, which is a major signaling mechanism for the pituitary adenylate cyclase-activating polypeptide (PACAP)-selective PAC1 receptor, in the regulation of guinea pig cardiac neuronal excitability. Forskolin (5-10 µM) increases excitability in ~60% of the cardiac neurons. The forskolin-mediated increase in excitability was considered related to cAMP regulation of a cyclic nucleotide gated channel or via protein kinase A (PKA)/ERK signaling, mechanisms that have been linked to PAC1 receptor activation. However, unlike PACAP mechanisms, forskolin enhancement of excitability was not significantly reduced by treatment with cesium to block currents through hyperpolarization-activated nonselective cation channels ( I h ) or by treatment with PD98059 to block MEK/ERK signaling. In contrast, treatment with the clathrin inhibitor Pitstop2 or the dynamin inhibitor dynasore eliminated the forskolin-induced increase in excitability; treatments with the inactive Pitstop analog or PP2 treatment to inhibit Src-mediated endocytosis mechanisms were ineffective. The PKA inhibitor KT5702 significantly suppressed the forskolin-induced change in excitability; further, KT5702 and Pitstop2 reduced the forskolin-stimulated MEK/ERK activation in cardiac neurons. Collectively, the present results suggest that forskolin activation of AC/cAMP/PKA signaling leads to the recruitment of clathrin/dynamin-dependent endosomal transduction cascades, including MEK/ERK signaling, and that endosomal signaling is the critical mechanism underlying the forskolin-induced increase in cardiac neuron excitability. Copyright © 2017 the American Physiological Society.

  18. Structural insights into biased G protein-coupled receptor signaling revealed by fluorescence spectroscopy.

    PubMed

    Rahmeh, Rita; Damian, Marjorie; Cottet, Martin; Orcel, Hélène; Mendre, Christiane; Durroux, Thierry; Sharma, K Shivaji; Durand, Grégory; Pucci, Bernard; Trinquet, Eric; Zwier, Jurriaan M; Deupi, Xavier; Bron, Patrick; Banères, Jean-Louis; Mouillac, Bernard; Granier, Sébastien

    2012-04-24

    G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters, representing the largest group of therapeutic targets. Recent studies show that some GPCRs signal through both G protein and arrestin pathways in a ligand-specific manner. Ligands that direct signaling through a specific pathway are known as biased ligands. The arginine-vasopressin type 2 receptor (V2R), a prototypical peptide-activated GPCR, is an ideal model system to investigate the structural basis of biased signaling. Although the native hormone arginine-vasopressin leads to activation of both the stimulatory G protein (Gs) for the adenylyl cyclase and arrestin pathways, synthetic ligands exhibit highly biased signaling through either Gs alone or arrestin alone. We used purified V2R stabilized in neutral amphipols and developed fluorescence-based assays to investigate the structural basis of biased signaling for the V2R. Our studies demonstrate that the Gs-biased agonist stabilizes a conformation that is distinct from that stabilized by the arrestin-biased agonists. This study provides unique insights into the structural mechanisms of GPCR activation by biased ligands that may be relevant to the design of pathway-biased drugs.

  19. Structural and Chemical Biology of Terpenoid Cyclases

    PubMed Central

    2017-01-01

    The year 2017 marks the twentieth anniversary of terpenoid cyclase structural biology: a trio of terpenoid cyclase structures reported together in 1997 were the first to set the foundation for understanding the enzymes largely responsible for the exquisite chemodiversity of more than 80000 terpenoid natural products. Terpenoid cyclases catalyze the most complex chemical reactions in biology, in that more than half of the substrate carbon atoms undergo changes in bonding and hybridization during a single enzyme-catalyzed cyclization reaction. The past two decades have witnessed structural, functional, and computational studies illuminating the modes of substrate activation that initiate the cyclization cascade, the management and manipulation of high-energy carbocation intermediates that propagate the cyclization cascade, and the chemical strategies that terminate the cyclization cascade. The role of the terpenoid cyclase as a template for catalysis is paramount to its function, and protein engineering can be used to reprogram the cyclization cascade to generate alternative and commercially important products. Here, I review key advances in terpenoid cyclase structural and chemical biology, focusing mainly on terpenoid cyclases and related prenyltransferases for which X-ray crystal structures have informed and advanced our understanding of enzyme structure and function. PMID:28841019

  20. The Orphan G Protein-coupled Receptor Gpr175 (Tpra40) Enhances Hedgehog Signaling by Modulating cAMP Levels.

    PubMed

    Singh, Jaskirat; Wen, Xiaohui; Scales, Suzie J

    2015-12-04

    The Hedgehog (Hh) signaling pathway plays an essential role in vertebrate embryonic tissue patterning of many developing organs. Signaling occurs predominantly in primary cilia and is initiated by the entry of the G protein-coupled receptor (GPCR)-like protein Smoothened into cilia and culminates in gene transcription via the Gli family of transcription factors upon their nuclear entry. Here we identify an orphan GPCR, Gpr175 (also known as Tpra1 or Tpra40: transmembrane protein, adipocyte associated 1 or of 40 kDa), which also localizes to primary cilia upon Hh stimulation and positively regulates Hh signaling. Interaction experiments place Gpr175 at the level of PKA and upstream of the Gαi component of heterotrimeric G proteins, which itself localizes to cilia and can modulate Hh signaling. Gpr175 or Gαi1 depletion leads to increases in cellular cAMP levels and in Gli3 processing into its repressor form. Thus we propose that Gpr175 coupled to Gαi1 normally functions to inhibit the production of cAMP by adenylyl cyclase upon Hh stimulation, thus maximizing signaling by turning off PKA activity and hence Gli3 repressor formation. Taken together our data suggest that Gpr175 is a novel positive regulator of the Hh signaling pathway. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Crystal structure of the Alpha subunit PAS domain from soluble guanylyl cyclase

    PubMed Central

    Purohit, Rahul; Weichsel, Andrzej; Montfort, William R

    2013-01-01

    Soluble guanylate cyclase (sGC) is a heterodimeric heme protein of ∼150 kDa and the primary nitric oxide receptor. Binding of NO stimulates cyclase activity, leading to regulation of cardiovascular physiology and providing attractive opportunities for drug discovery. How sGC is stimulated and where candidate drugs bind remains unknown. The α and β sGC chains are each composed of Heme-Nitric Oxide Oxygen (H-NOX), Per-ARNT-Sim (PAS), coiled-coil and cyclase domains. Here, we present the crystal structure of the α1 PAS domain to 1.8 Å resolution. The structure reveals the binding surfaces of importance to heterodimer function, particularly with respect to regulating NO binding to heme in the β1 H-NOX domain. It also reveals a small internal cavity that may serve to bind ligands or participate in signal transduction. PMID:23934793

  2. Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I.

    PubMed

    Lama, Lodoe; Ryan, Kevin

    2016-01-01

    Many high-throughput small RNA next-generation sequencing protocols use 5' preadenylylated DNA oligonucleotide adapters during cDNA library preparation. Preadenylylation of the DNA adapter's 5' end frees from ATP-dependence the ligation of the adapter to RNA collections, thereby avoiding ATP-dependent side reactions. However, preadenylylation of the DNA adapters can be costly and difficult. The currently available method for chemical adenylylation of DNA adapters is inefficient and uses techniques not typically practiced in laboratories profiling cellular RNA expression. An alternative enzymatic method using a commercial RNA ligase was recently introduced, but this enzyme works best as a stoichiometric adenylylating reagent rather than a catalyst and can therefore prove costly when several variant adapters are needed or during scale-up or high-throughput adenylylation procedures. Here, we describe a simple, scalable, and highly efficient method for the 5' adenylylation of DNA oligonucleotides using the thermostable RNA ligase 1 from bacteriophage TS2126. Adapters with 3' blocking groups are adenylylated at >95% yield at catalytic enzyme-to-adapter ratios and need not be gel purified before ligation to RNA acceptors. Experimental conditions are also reported that enable DNA adapters with free 3' ends to be 5' adenylylated at >90% efficiency. © 2015 Lama and Ryan; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  3. Adenylylation of mycobacterial Glnk (PII) protein is induced by nitrogen limitation

    PubMed Central

    Williams, Kerstin J.; Bennett, Mark H.; Barton, Geraint R.; Jenkins, Victoria A.; Robertson, Brian D.

    2013-01-01

    Summary PII proteins are pivotal regulators of nitrogen metabolism in most prokaryotes, controlling the activities of many targets, including nitrogen assimilation enzymes, two component regulatory systems and ammonium transport proteins. Escherichia coli contains two PII-like proteins, PII (product of glnB) and GlnK, both of which are uridylylated under nitrogen limitation at a conserved Tyrosine-51 residue by GlnD (a uridylyl transferase). PII-uridylylation in E. coli controls glutamine synthetase (GS) adenylylation by GlnE and mediates the NtrB/C transcriptomic response. Mycobacteria contain only one PII protein (GlnK) which in environmental Actinomycetales is adenylylated by GlnD under nitrogen limitation. However in mycobacteria, neither the type of GlnK (PII) covalent modification nor its precise role under nitrogen limitation is known. In this study, we used LC-Tandem MS to analyse the modification state of mycobacterial GlnK (PII), and demonstrate that during nitrogen limitation GlnK from both non-pathogenic Mycobacterium smegmatis and pathogenic Mycobacterium tuberculosis is adenylylated at the Tyrosine-51 residue; we also show that GlnD is the adenylyl transferase enzyme responsible. Further analysis shows that in contrast to E. coli, GlnK (PII) adenylylation in M. tuberculosis does not regulate GS adenylylation, nor does it mediate the transcriptomic response to nitrogen limitation. PMID:23352854

  4. Molecular Physiology of Membrane Guanylyl Cyclase Receptors.

    PubMed

    Kuhn, Michaela

    2016-04-01

    cGMP controls many cellular functions ranging from growth, viability, and differentiation to contractility, secretion, and ion transport. The mammalian genome encodes seven transmembrane guanylyl cyclases (GCs), GC-A to GC-G, which mainly modulate submembrane cGMP microdomains. These GCs share a unique topology comprising an extracellular domain, a short transmembrane region, and an intracellular COOH-terminal catalytic (cGMP synthesizing) region. GC-A mediates the endocrine effects of atrial and B-type natriuretic peptides regulating arterial blood pressure/volume and energy balance. GC-B is activated by C-type natriuretic peptide, stimulating endochondral ossification in autocrine way. GC-C mediates the paracrine effects of guanylins on intestinal ion transport and epithelial turnover. GC-E and GC-F are expressed in photoreceptor cells of the retina, and their activation by intracellular Ca(2+)-regulated proteins is essential for vision. Finally, in the rodent system two olfactorial GCs, GC-D and GC-G, are activated by low concentrations of CO2and by peptidergic (guanylins) and nonpeptidergic odorants as well as by coolness, which has implications for social behaviors. In the past years advances in human and mouse genetics as well as the development of sensitive biosensors monitoring the spatiotemporal dynamics of cGMP in living cells have provided novel relevant information about this receptor family. This increased our understanding of the mechanisms of signal transduction, regulation, and (dys)function of the membrane GCs, clarified their relevance for genetic and acquired diseases and, importantly, has revealed novel targets for therapies. The present review aims to illustrate these different features of membrane GCs and the main open questions in this field. Copyright © 2016 the American Physiological Society.

  5. Effects of Lubrol detergents on adenylate cyclases.

    PubMed

    Bär, H P; Kulshrestha, S

    1975-04-01

    The nonionic detergent Lubrol WX showed diverse, concentration-dependent effects onbasal and stimulated adenylate cyclases. Above concentrations of 0.001-0.01% Lubrol WX, the basal activity of cyclase from Ehrlich ascites cells was inhibed about 50%, and that from rat fat cells was doubled. In both cases, hormonal sensitivity was lost at 0.01%. These effects were reversed upon dilution of the detergent. It is suggested that solubilization of adenylate cyclases at such low concentrations of Lubrol should be attempted since it is conceivable that loss of hormone sensitivity may then be reversible. Different Lubrol-type detergents may also offer centain advantages, since Lubrol PX effects were not identical with those of Lubrol WX.

  6. Bifunctional Homodimeric Triokinase/FMN Cyclase

    PubMed Central

    Rodrigues, Joaquim Rui; Couto, Ana; Cabezas, Alicia; Pinto, Rosa María; Ribeiro, João Meireles; Canales, José; Costas, María Jesús; Cameselle, José Carlos

    2014-01-01

    Mammalian triokinase, which phosphorylates exogenous dihydroxyacetone and fructose-derived glyceraldehyde, is neither molecularly identified nor firmly associated to an encoding gene. Human FMN cyclase, which splits FAD and other ribonucleoside diphosphate-X compounds to ribonucleoside monophosphate and cyclic X-phosphodiester, is identical to a DAK-encoded dihydroxyacetone kinase. This bifunctional protein was identified as triokinase. It was modeled as a homodimer of two-domain (K and L) subunits. Active centers lie between K1 and L2 or K2 and L1: dihydroxyacetone binds K and ATP binds L in different subunits too distant (≈14 Å) for phosphoryl transfer. FAD docked to the ATP site with ribityl 4′-OH in a possible near-attack conformation for cyclase activity. Reciprocal inhibition between kinase and cyclase reactants confirmed substrate site locations. The differential roles of protein domains were supported by their individual expression: K was inactive, and L displayed cyclase but not kinase activity. The importance of domain mobility for the kinase activity of dimeric triokinase was highlighted by molecular dynamics simulations: ATP approached dihydroxyacetone at distances below 5 Å in near-attack conformation. Based upon structure, docking, and molecular dynamics simulations, relevant residues were mutated to alanine, and kcat and Km were assayed whenever kinase and/or cyclase activity was conserved. The results supported the roles of Thr112 (hydrogen bonding of ATP adenine to K in the closed active center), His221 (covalent anchoring of dihydroxyacetone to K), Asp401 and Asp403 (metal coordination to L), and Asp556 (hydrogen bonding of ATP or FAD ribose to L domain). Interestingly, the His221 point mutant acted specifically as a cyclase without kinase activity. PMID:24569995

  7. Differential inhibition of adenylylated and deadenylylated forms of M. tuberculosis glutamine synthetase as a drug discovery platform

    PubMed Central

    Theron, A.; Roth, R. L.; Hoppe, H.; Parkinson, C.; van der Westhuyzen, C. W.; Stoychev, S.; Wiid, I.; Pietersen, R. D.; Baker, B.

    2017-01-01

    Glutamine synthetase is a ubiquitous central enzyme in nitrogen metabolism that is controlled by up to four regulatory mechanisms, including adenylylation of some or all of the twelve subunits by adenylyl transferase. It is considered a potential therapeutic target for the treatment of tuberculosis, being essential for the growth of Mycobacterium tuberculosis, and is found extracellularly only in the pathogenic Mycobacterium strains. Human glutamine synthetase is not regulated by the adenylylation mechanism, so the adenylylated form of bacterial glutamine synthetase is of particular interest. Previously published reports show that, when M. tuberculosis glutamine synthetase is expressed in Escherichia coli, the E. coli adenylyl transferase does not optimally adenylylate the M. tuberculosis glutamine synthetase. Here, we demonstrate the production of soluble adenylylated M. tuberulosis glutamine synthetase in E. coli by the co-expression of M. tuberculosis glutamine synthetase and M. tuberculosis adenylyl transferase. The differential inhibition of adenylylated M. tuberulosis glutamine synthetase and deadenylylated M. tuberulosis glutamine synthetase by ATP based scaffold inhibitors are reported. Compounds selected on the basis of their enzyme inhibition were also shown to inhibit M. tuberculosis in the BACTEC 460TB™ assay as well as the intracellular inhibition of M. tuberculosis in a mouse bone-marrow derived macrophage assay. PMID:28972974

  8. CD38-dependent ADP-ribosyl cyclase activity in developing and adult mouse brain.

    PubMed Central

    Ceni, Claire; Pochon, Nathalie; Brun, Virginie; Muller-Steffner, Hélène; Andrieux, Annie; Grunwald, Didier; Schuber, Francis; De Waard, Michel; Lund, Frances; Villaz, Michel; Moutin, Marie-Jo

    2003-01-01

    CD38 is a transmembrane glycoprotein that is expressed in many tissues throughout the body. In addition to its major NAD+-glycohydrolase activity, CD38 is also able to synthesize cyclic ADP-ribose, an endogenous calcium-regulating molecule, from NAD+. In the present study, we have compared ADP-ribosyl cyclase and NAD+-glycohydrolase activities in protein extracts of brains from developing and adult wild-type and Cd38 -/- mice. In extracts from wild-type brain, cyclase activity was detected spectrofluorimetrically, using nicotinamide-guanine dinucleotide as a substrate (GDP-ribosyl cyclase activity), as early as embryonic day 15. The level of cyclase activity was similar in the neonate brain (postnatal day 1) and then increased greatly in the adult brain. Using [14C]NAD+ as a substrate and HPLC analysis, we found that ADP-ribose is the major product formed in the brain at all developmental stages. Under the same experimental conditions, neither NAD+-glycohydrolase nor GDP-ribosyl cyclase activity could be detected in extracts of brains from developing or adult Cd38 -/- mice, demonstrating that CD38 is the predominant constitutive enzyme endowed with these activities in brain at all developmental stages. The activity measurements correlated with the level of CD38 transcripts present in the brains of developing and adult wild-type mice. Using confocal microscopy we showed, in primary cultures of hippocampal cells, that CD38 is expressed by both neurons and glial cells, and is enriched in neuronal perikarya. Intracellular NAD+-glycohydrolase activity was measured in hippocampal cell cultures, and CD38-dependent cyclase activity was higher in brain fractions enriched in intracellular membranes. Taken together, these results lead us to speculate that CD38 might have an intracellular location in neural cells in addition to its plasma membrane location, and may play an important role in intracellular cyclic ADP-ribose-mediated calcium signalling in brain tissue. PMID

  9. FABP4 is secreted from adipocytes by adenyl cyclase-PKA- and guanylyl cyclase-PKG-dependent lipolytic mechanisms.

    PubMed

    Mita, Tomohiro; Furuhashi, Masato; Hiramitsu, Shinya; Ishii, Junnichi; Hoshina, Kyoko; Ishimura, Shutaro; Fuseya, Takahiro; Watanabe, Yuki; Tanaka, Marenao; Ohno, Kohei; Akasaka, Hiroshi; Ohnishi, Hirofumi; Yoshida, Hideaki; Saitoh, Shigeyuki; Shimamoto, Kazuaki; Miura, Tetsuji

    2015-02-01

    Fatty acid-binding protein 4 (FABP4) is expressed in adipocytes, and elevated plasma FABP4 level is associated with obesity-mediated metabolic phenotype. Postprandial regulation and secretory signaling of FABP4 has been investigated. Time courses of FABP4 levels were examined during an oral glucose tolerance test (OGTT; n=53) or a high-fat test meal eating (n=35). Effects of activators and inhibitors of adenyl cyclase (AC)-protein kinase A (PKA) signaling and guanylyl cyclase (GC)-protein kinase G (PKG) signaling on FABP4 secretion from mouse 3T3-L1 adipocytes were investigated. FABP4 level significantly declined after the OGTT or a high-fat meal eating, while insulin level was increased. Treatment with low and high glucose concentration or palmitate for 2 h did not affect FABP4 secretion from 3T3-L1 adipocytes. FABP4 secretion was increased by stimulation of lipolysis using isoproterenol, a β3 -adrenoceptor agonist (CL316243), forskolin, dibutyryl-cAMP and atrial natriuretic peptide, and the induced FABP4 secretion was suppressed by insulin or an inhibitor of PKA (H-89), PKG (KT5823) or hormone sensitive lipase (CAY10499). FABP4 is secreted from adipocytes in association with lipolysis regulated by AC-PKA- and GC-PKG-mediated signal pathways. Plasma FABP4 level declines postprandially, and suppression of FABP4 secretion by insulin-induced anti-lipolytic signaling may be involved in this decline in FABP4 level. © 2014 The Obesity Society.

  10. Distinct intracellular sAC-cAMP domains regulate ER Ca2+ signaling and OXPHOS function.

    PubMed

    Valsecchi, Federica; Konrad, Csaba; D'Aurelio, Marilena; Ramos-Espiritu, Lavoisier S; Stepanova, Anna; Burstein, Suzanne R; Galkin, Alexander; Magranè, Jordi; Starkov, Anatoly; Buck, Jochen; Levin, Lonny R; Manfredi, Giovanni

    2017-11-01

    cAMP regulates a wide variety of physiological functions in mammals. This single second messenger can regulate multiple, seemingly disparate functions within independently regulated cell compartments. We have previously identified one such compartment inside the matrix of the mitochondria, where soluble adenylyl cyclase (sAC) regulates oxidative phosphorylation (OXPHOS). We now show that sAC knockout fibroblasts have a defect in OXPHOS activity and attempt to compensate for this defect by increasing OXPHOS proteins. Importantly, sAC knockout cells also exhibit decreased probability of endoplasmic reticulum (ER) Ca 2+ release associated with diminished phosphorylation of the inositol 3-phosphate receptor. Restoring sAC expression exclusively in the mitochondrial matrix rescues OXPHOS activity and reduces mitochondrial biogenesis, indicating that these phenotypes are regulated by intramitochondrial sAC. In contrast, Ca 2+ release from the ER is only rescued when sAC expression is restored throughout the cell. Thus, we show that functionally distinct, sAC-defined, intracellular cAMP signaling domains regulate metabolism and Ca 2+ signaling. © 2017. Published by The Company of Biologists Ltd.

  11. cAMP signaling mediates behavioral flexibility and consolidation of social status in Drosophila aggression.

    PubMed

    Chouhan, Nitin Singh; Mohan, Krithika; Ghose, Aurnab

    2017-12-01

    Social rituals, such as male-male aggression in Drosophila , are often stereotyped and the component behavioral patterns modular. The likelihood of transition from one behavioral pattern to another is malleable by experience and confers flexibility to the behavioral repertoire. Experience-dependent modification of innate aggressive behavior in flies alters fighting strategies during fights and establishes dominant-subordinate relationships. Dominance hierarchies resulting from agonistic encounters are consolidated to longer-lasting, social-status-dependent behavioral modifications, resulting in a robust loser effect. We showed that cAMP dynamics regulated by the calcium-calmodulin-dependent adenylyl cyclase, Rut, and the cAMP phosphodiesterase, Dnc, but not the Amn gene product, in specific neuronal groups of the mushroom body and central complex, mediate behavioral plasticity necessary to establish dominant-subordinate relationships. rut and dnc mutant flies were unable to alter fighting strategies and establish dominance relationships during agonistic interactions. This real-time flexibility during a fight was independent of changes in aggression levels. Longer-term consolidation of social status in the form of a loser effect, however, required additional Amn -dependent inputs to cAMP signaling and involved a circuit-level association between the α/β and γ neurons of the mushroom body. Our findings implicate cAMP signaling in mediating the plasticity of behavioral patterns in aggressive behavior and in the generation of a temporally stable memory trace that manifests as a loser effect. © 2017. Published by The Company of Biologists Ltd.

  12. Bordetella Adenylate Cyclase-Hemolysin Toxins

    PubMed Central

    Guiso, Nicole

    2017-01-01

    Adenylate cyclase-hemolysin toxin is secreted and produced by three classical species of the genus Bordetella: Bordetella pertussis, B. parapertussis and B. bronchiseptica. This toxin has several properties such as: (i) adenylate cyclase activity, enhanced after interaction with the eukaryotic protein, calmodulin; (ii) a pore-forming activity; (iii) an invasive activity. It plays an important role in the pathogenesis of these Bordetella species responsible for whooping cough in humans or persistent respiratory infections in mammals, by modulating host immune responses. In contrast with other Bordetella toxins or adhesins, lack of (or very low polymorphism) is observed in the structural gene encoding this toxin, supporting its importance as well as a potential role as a vaccine antigen against whooping cough. In this article, an overview of the investigations undertaken on this toxin is presented. PMID:28892012

  13. Soluble guanylate cyclase generation of cGMP regulates migration of MGE neurons.

    PubMed

    Mandal, Shyamali; Stanco, Amelia; Buys, Emmanuel S; Enikolopov, Grigori; Rubenstein, John L R

    2013-10-23

    Here we have provided evidence that nitric oxide-cyclic GMP (NO-cGMP) signaling regulates neurite length and migration of immature neurons derived from the medial ganglionic eminence (MGE). Dlx1/2(-/-) and Lhx6(-/-) mouse mutants, which exhibit MGE interneuron migration defects, have reduced expression of the gene encoding the α subunit of a soluble guanylate cyclase (Gucy1A3). Furthermore, Dlx1/2(-/-) mouse mutants have reduced expression of NO synthase 1 (NOS1). Gucy1A3(-/-) mice have a transient reduction in cortical interneuron number. Pharmacological inhibition of soluble guanylate cyclase and NOS activity rapidly induces neurite retraction of MGE cells in vitro and in slice culture and robustly inhibits cell migration from the MGE and caudal ganglionic eminence. We provide evidence that these cellular phenotypes are mediated by activation of the Rho signaling pathway and inhibition of myosin light chain phosphatase activity.

  14. Glucose and cyclic adenosine monophosphate stimulate activities of adenylate cyclase and guanylate cyclase of Tetrahymena pyriformis infusoria.

    PubMed

    Shpakov, A O; Derkach, K V; Uspenskaya, Z I

    2012-02-01

    The sensitivities of cyclase enzymes adenylate cyclase and guanylate cyclase to glucose and extracellular cAMP were studied in Tetrahymena pyriformis infusoria. Glucose effectively stimulated activities of both cyclase enzymes, while cAMP more effectively stimulated adenylate cyclase. It was shown that [6-(14)C]glucose specifically bound to Tetrahymena pyriformis infusoria at dissociation constant (K(D)) and number of binding sites (B(max)) 43 nM and 7.53 fmol glucose per 100,000 cells and [8-(3)H]cAMP bound at 19 nM and 4.46 fmol cAMP per 100,000 cells, respectively. Hence, glucose and cAMP specifically bound to Tetrahymena pyriformis cells and stimulated activities of cyclases in these infusoria.

  15. CCK receptors-related signaling involved in nitric oxide production caused by gastrin 17 in porcine coronary endothelial cells.

    PubMed

    Grossini, Elena; Caimmi, Philippe; Molinari, Claudio; Uberti, Francesca; Mary, David; Vacca, Giovanni

    2012-03-05

    In anesthetized pigs gastrin-17 increased coronary blood flow through CCK1/CCK2 receptors and β(2)-adrenoceptors-related nitric oxide (NO) release. Since the intracellular pathway has not been investigated the purpose of this study was to examine in coronary endothelial cells the CCK1/CCK2 receptors-related signaling involved in the effects of gastrin-17 on NO release. Gastrin-17 caused a concentration-dependent increase of NO production (17.3-62.6%; p<0.05), which was augmented by CCK1/CCK2 receptors agonists (p<0.05). The effect of gastrin-17 was amplified by the adenylyl-cyclase activator and β(2)-adrenoceptors agonist (p<0.05), abolished by cAMP/PKA and β(2)-adrenoceptors and CCK1/CCK2 receptors blockers, and reduced by PLC/PKC inhibitor. Finally, Western-blot revealed the preferential involvement of PKA vs. PKC as downstream effectors of CCK1/CCK2 receptors activation leading to Akt, ERK, p38 and endothelial NOS (eNOS) phosphorylation. In conclusion, in coronary endothelial cells, gastrin-17 induced eNOS-dependent NO production through CCK1/CCK2 receptors- and β(2)-adrenoceptors-related pathway. The intracellular signaling involved a preferential PKA pathway over PKC. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  16. Somatostatin Signaling in Neuronal Cilia Is Criticalfor Object Recognition Memory

    PubMed Central

    Einstein, Emily B.; Patterson, Carlyn A.; Hon, Beverly J.; Regan, Kathleen A.; Reddi, Jyoti; Melnikoff, David E.; Mateer, Marcus J.; Schulz, Stefan; Johnson, Brian N.

    2010-01-01

    Most neurons possess a single, nonmotile cilium that projects out from the cell surface. These microtubule-based organelles are important in brain development and neurogenesis; however, their function in mature neurons is unknown. Cilia express a complement of proteins distinct from other neuronal compartments, one of which is the somatostatin receptor subtype SST3. We show here that SST3 is critical for object recognition memory in mice. sst3 knock-out mice are severely impaired in discriminating novel objects, whereas they retain normal memory for object location. Further, systemic injection of an SST3 antagonist (ACQ090) disrupts recall of familiar objects in wild-type mice. To examine mechanisms of SST3, we tested synaptic plasticity in CA1 hippocampus. Electrically evoked long-term potentiation (LTP) was normal in sst3 knock-out mice, while adenylyl cyclase/cAMP-mediated LTP was impaired. The SST3 antagonist also disrupted cAMP-mediated LTP. Basal cAMP levels in hippocampal lysate were reduced in sst3 knock-out mice compared with wild-type mice, while the forskolin-induced increase in cAMP levels was normal. The SST3 antagonist inhibited forskolin-stimulated cAMP increases, whereas the SST3 agonist L-796,778 increased basal cAMP levels in hippocampal slices but not hippocampal lysate. Our results show that somatostatin signaling in neuronal cilia is critical for recognition memory and suggest that the cAMP pathway is a conserved signaling motif in cilia. Neuronal cilia therefore represent a novel nonsynaptic compartment crucial for signaling involved in a specific form of synaptic plasticity and in novelty detection. PMID:20335466

  17. The size of adenylate cyclase and guanylate cyclase from the rat renal medulla.

    PubMed

    Neer, E J

    1976-01-01

    The size distribution of adenylate cyclase from the rat renal medulla solubilized with the nonionic detergents Triton X-100 and Lubrol PX was determined by gel filtration and by centrifugation in sucrose density gradients made up in H2O or D2O. The physical parameters of the predominant form in Triton X-100 are s20,w, 5.9S; Strokes radius, 62 A; partial specific volume (v), 0.74 ml/g; mass, 159,000 daltons; f/f0, 1.6; axial ratio (prolate ellipsoid), 11. For the minor form the values are: s20w, 3.0; Stokes radius, 28 A; mass, 38,000 daltons; f/f0, 1.2. The corresponding values determined in Lubrol PX are similar. The value for V for the enzyme indicates that it binds less than 0.2 mg detergent/mg protein. Since interactions with detergents probably substitute for interactions with lipids and hydrophobic amino acid side chains, these findings suggest that no more than 5% of the surface of adenylate cyclase is involved in hydrophobic interactions with other membrane components. Thus, most of the mass of the enzyme is not deeply embedded in the lipid bilayer of the plasma membrane. Similar studies have been performed on the soluble guanylate cyclase of the rat renal medulla. In the absence of detergent, the molecular properties of this enzyme are: s20w, 6.3S; Stokes radius, 54 A, V, 0.75 ml/g; mass, 154,000 daltons f/f0, 1.4; Axial ratio, 7. The addition of 0.1% Lubrol PX to this soluble enzyme increases it activity two- to fourfold and changes the physical properties to: s20,w, 5.5S; Stokes radius, 62 A; V, 0.74 ml/g; mass, 148,000 daltons, f/f0, 1.6; axial ratio, 11. These results show that Lubrol PX activates the enzyme by causing a conformational change with unfolding on the polypeptide chain. Guanylate cyclase from the particulate cell fraction can be solubilized with Lubrol PX but has properties quite different from those of the enzyme in the soluble cell fraction. It is a heterogeneous aggregate with s20,w, 10S; Stokes radius, 65 A; mass about 300,000 daltons

  18. Voluntary running depreciates the requirement of Ca2+-stimulated cAMP signaling in synaptic potentiation and memory formation

    PubMed Central

    Zheng, Fei; Zhang, Ming; Ding, Qi; Sethna, Ferzin; Yan, Lily; Moon, Changjong; Yang, Miyoung

    2016-01-01

    Mental health and cognitive functions are influenced by both genetic and environmental factors. Although having active lifestyle with physical exercise improves learning and memory, how it interacts with the specific key molecular regulators of synaptic plasticity is largely unknown. Here, we examined the effects of voluntary running on long-term potentiation (LTP) and memory formation in mice lacking type 1 adenylyl cyclase (AC1), a neurospecific synaptic enzyme that contributes to Ca2+-stimulated cAMP production. Following 1 mo of voluntary running-wheel exercise, the impaired LTP and object recognition memory in AC1 knockout (KO) mice were significantly attenuated. Running up-regulated exon II mRNA level of BDNF (brain-derived neurotrophic factor), though it failed to increase exon I and IV mRNAs in the hippocampus of AC1 KO mice. Intrahippocampal infusion of recombinant BDNF was sufficient to rescue LTP and object recognition memory defects in AC1 KO mice. Therefore, voluntary running and exogenous BDNF application overcome the defective Ca2+-stimulated cAMP signaling. Our results also demonstrate that alteration in Ca2+-stimulated cAMP can affect the molecular outcome of physical exercise. PMID:27421897

  19. Short- and long-term memory in Drosophila require cAMP signaling in distinct neuron types.

    PubMed

    Blum, Allison L; Li, Wanhe; Cressy, Mike; Dubnau, Josh

    2009-08-25

    A common feature of memory and its underlying synaptic plasticity is that each can be dissected into short-lived forms involving modification or trafficking of existing proteins and long-term forms that require new gene expression. An underlying assumption of this cellular view of memory consolidation is that these different mechanisms occur within a single neuron. At the neuroanatomical level, however, different temporal stages of memory can engage distinct neural circuits, a notion that has not been conceptually integrated with the cellular view. Here, we investigated this issue in the context of aversive Pavlovian olfactory memory in Drosophila. Previous studies have demonstrated a central role for cAMP signaling in the mushroom body (MB). The Ca(2+)-responsive adenylyl cyclase RUTABAGA is believed to be a coincidence detector in gamma neurons, one of the three principle classes of MB Kenyon cells. We were able to separately restore short-term or long-term memory to a rutabaga mutant with expression of rutabaga in different subsets of MB neurons. Our findings suggest a model in which the learning experience initiates two parallel associations: a short-lived trace in MB gamma neurons, and a long-lived trace in alpha/beta neurons.

  20. Inhibition of Heat-Stable Toxin-Induced Intestinal Salt and Water Secretion by a Novel Class of Guanylyl Cyclase C Inhibitors.

    PubMed

    Bijvelds, Marcel J C; Loos, Michaela; Bronsveld, Inez; Hellemans, Ann; Bongartz, Jean-Pierre; Ver Donck, Luc; Cox, Eric; de Jonge, Hugo R; Schuurkes, Jan A J; De Maeyer, Joris H

    2015-12-01

    Many enterotoxigenic Escherichia coli strains produce the heat-stable toxin, STa, which, by activation of the intestinal receptor-enzyme guanylyl cyclase (GC) C, triggers an acute, watery diarrhea. We set out to identify GCC inhibitors that may be of benefit for the treatment of infectious diarrheal disease. Compounds that inhibit STa-induced cyclic guanosine 3',5'-monophosphate (cGMP) production were selected by performing cyclase assays on cells and membranes containing GCC, or the related GCA. The effect of leads on STa/GCC-dependent activation of the cystic fibrosis transmembrane conductance regulator anion channel was investigated in T84 cells, and in porcine and human intestinal tissue. Their effect on STa-provoked fluid transport was assessed in ligated intestinal loops in piglets. Four N-2-(propylamino)-6-phenylpyrimidin-4-one-substituted piperidines were shown to inhibit GCC-mediated cellular cGMP production. The half maximal inhibitory concentrations were ≤ 5 × 10(-7) mol/L, whereas they were >10 times higher for GCA. In T84 monolayers, these leads blocked STa/GCC-dependent, but not forskolin/adenylyl cyclase-dependent, cystic fibrosis transmembrane conductance regulator activity. GCC inhibition reduced STa-provoked anion secretion in pig jejunal tissue, and fluid retention and cGMP levels in STa-exposed loops. These GCC inhibitors blocked STa-provoked anion secretion in rectal biopsy specimens. We have identified a novel class of GCC inhibitors that may form the basis for development of future therapeutics for (infectious) diarrheal disease. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  1. β2-Adrenergic receptor activation mobilizes intracellular calcium via a non-canonical cAMP-independent signaling pathway.

    PubMed

    Galaz-Montoya, Monica; Wright, Sara J; Rodriguez, Gustavo J; Lichtarge, Olivier; Wensel, Theodore G

    2017-06-16

    Beta adrenergic receptors (βARs) are G-protein-coupled receptors essential for physiological responses to the hormones/neurotransmitters epinephrine and norepinephrine which are found in the nervous system and throughout the body. They are the targets of numerous widely used drugs, especially in the case of the most extensively studied βAR, β 2 AR, whose ligands are used for asthma and cardiovascular disease. βARs signal through Gα s G-proteins and via activation of adenylyl cyclase and cAMP-dependent protein kinase, but some alternative downstream pathways have also been proposed that could be important for understanding normal physiological functioning of βAR signaling and its disruption in disease. Using fluorescence-based Ca 2+ flux assays combined with pharmacology and gene knock-out methods, we discovered a previously unrecognized endogenous pathway in HEK-293 cells whereby β 2 AR activation leads to robust Ca 2+ mobilization from intracellular stores via activation of phospholipase C and opening of inositol trisphosphate (InsP 3 ) receptors. This pathway did not involve cAMP, Gα s , or Gα i or the participation of the other members of the canonical β 2 AR signaling cascade and, therefore, constitutes a novel signaling mechanism for this receptor. This newly uncovered mechanism for Ca 2+ mobilization by β 2 AR has broad implications for adrenergic signaling, cross-talk with other signaling pathways, and the effects of βAR-directed drugs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Cyclic adenosine monophosphate metabolism in synaptic growth, strength, and precision: neural and behavioral phenotype-specific counterbalancing effects between dnc phosphodiesterase and rut adenylyl cyclase mutations.

    PubMed

    Ueda, Atsushi; Wu, Chun-Fang

    2012-03-01

    Two classic learning mutants in Drosophila, rutabaga (rut) and dunce (dnc), are defective in cyclic adenosine monophosphate (cAMP) synthesis and degradation, respectively, exhibiting a variety of neuronal and behavioral defects. We ask how the opposing effects of these mutations on cAMP levels modify subsets of phenotypes, and whether any specific phenotypes could be ameliorated by biochemical counter balancing effects in dnc rut double mutants. Our study at larval neuromuscular junctions (NMJs) demonstrates that dnc mutations caused severe defects in nerve terminal morphology, characterized by unusually large synaptic boutons and aberrant innervation patterns. Interestingly, a counterbalancing effect led to rescue of the aberrant innervation patterns but the enlarged boutons in dnc rut double mutant remained as extreme as those in dnc. In contrast to dnc, rut mutations strongly affect synaptic transmission. Focal loose-patch recording data accumulated over 4 years suggest that synaptic currents in rut boutons were characterized by unusually large temporal dispersion and a seasonal variation in the amount of transmitter release, with diminished synaptic currents in summer months. Experiments with different rearing temperatures revealed that high temperature (29-30°C) decreased synaptic transmission in rut, but did not alter dnc and wild-type (WT). Importantly, the large temporal dispersion and abnormal temperature dependence of synaptic transmission, characteristic of rut, still persisted in dnc rut double mutants. To interpret these results in a proper perspective, we reviewed previously documented differential effects of dnc and rut mutations and their genetic interactions in double mutants on a variety of physiological and behavioral phenotypes. The cases of rescue in double mutants are associated with gradual developmental and maintenance processes whereas many behavioral and physiological manifestations on faster time scales could not be rescued. We discuss factors that could contribute to the effectiveness of counterbalancing interactions between dnc and rut mutations for phenotypic rescue.

  3. The effects of sex and neonatal stress on pituitary adenylate cyclase-activating peptide expression.

    PubMed

    Mosca, E V; Rousseau, J P; Gulemetova, R; Kinkead, R; Wilson, R J A

    2015-02-01

    What is the central question of this study? Does sex or neonatal stress affect the expression of pituitary adenylate cyclase-activating peptide or its receptors? What is the main finding and its importance? Neonatal-maternal separation stress has little long-lasting effect on the expression of pituitary adenylate cyclase-activating peptide or its receptors, but sex differences exist in these genes between males and females at baseline. Sex differences in classic stress hormones have been studied in depth, but pituitary adenylate cyclase-activating peptide (PACAP), recently identified as playing a critical role in the stress axes, has not. Here we studied whether baseline levels of PACAP differ between sexes in various stress-related tissues and whether neonatal-maternal separation stress has a sex-dependent effect on PACAP gene expression in stress pathways. Using quantitative RT-PCR, we found sex differences in PACAP and PACAP receptor gene expression in several respiratory and/or stress-related tissues, while neonatal-maternal separation stress did little to affect PACAP signalling in adult animals. We propose that sex differences in PACAP expression are likely to contribute to differences between males and females in responses to stress. © 2015 The Authors. Experimental Physiology © 2015 The Physiological Society.

  4. The isoenzyme of glutaminyl cyclase is an important regulator of monocyte infiltration under inflammatory conditions

    PubMed Central

    Cynis, Holger; Hoffmann, Torsten; Friedrich, Daniel; Kehlen, Astrid; Gans, Kathrin; Kleinschmidt, Martin; Rahfeld, Jens-Ulrich; Wolf, Raik; Wermann, Michael; Stephan, Anett; Haegele, Monique; Sedlmeier, Reinhard; Graubner, Sigrid; Jagla, Wolfgang; Müller, Anke; Eichentopf, Rico; Heiser, Ulrich; Seifert, Franziska; Quax, Paul H A; de Vries, Margreet R; Hesse, Isabel; Trautwein, Daniela; Wollert, Ulrich; Berg, Sabine; Freyse, Ernst-Joachim; Schilling, Stephan; Demuth, Hans-Ulrich

    2011-01-01

    Acute and chronic inflammatory disorders are characterized by detrimental cytokine and chemokine expression. Frequently, the chemotactic activity of cytokines depends on a modified N-terminus of the polypeptide. Among those, the N-terminus of monocyte chemoattractant protein 1 (CCL2 and MCP-1) is modified to a pyroglutamate (pE-) residue protecting against degradation in vivo. Here, we show that the N-terminal pE-formation depends on glutaminyl cyclase activity. The pE-residue increases stability against N-terminal degradation by aminopeptidases and improves receptor activation and signal transduction in vitro. Genetic ablation of the glutaminyl cyclase iso-enzymes QC (QPCT) or isoQC (QPCTL) revealed a major role of isoQC for pE1-CCL2 formation and monocyte infiltration. Consistently, administration of QC-inhibitors in inflammatory models, such as thioglycollate-induced peritonitis reduced monocyte infiltration. The pharmacologic efficacy of QC/isoQC-inhibition was assessed in accelerated atherosclerosis in ApoE3*Leiden mice, showing attenuated atherosclerotic pathology following chronic oral treatment. Current strategies targeting CCL2 are mainly based on antibodies or spiegelmers. The application of small, orally available inhibitors of glutaminyl cyclases represents an alternative therapeutic strategy to treat CCL2-driven disorders such as atherosclerosis/restenosis and fibrosis. PMID:21774078

  5. β1-adrenergic receptors activate two distinct signaling pathways in striatal neurons

    PubMed Central

    Meitzen, John; Luoma, Jessie I.; Stern, Christopher M.; Mermelstein, Paul G.

    2010-01-01

    Monoamine action in the dorsal striatum and nucleus accumbens plays essential roles in striatal physiology. Although research often focuses on dopamine and its receptors, norepinephrine and adrenergic receptors are also crucial in regulating striatal function. While noradrenergic neurotransmission has been identified in the striatum, little is known regarding the signaling pathways activated by β-adrenergic receptors in this brain region. Using cultured striatal neurons, we characterized a novel signaling pathway by which activation of β1-adrenergic receptors leads to the rapid phosphorylation of cAMP Response Element Binding Protein (CREB), a transcription-factor implicated as a molecular switch underlying long-term changes in brain function. Norepinephrine-mediated CREB phosphorylation requires β1-adrenergic receptor stimulation of a receptor tyrosine kinase, ultimately leading to the activation of a Ras/Raf/MEK/MAPK/MSK signaling pathway. Activation of β1-adrenergic receptors also induces CRE-dependent transcription and increased c-fos expression. In addition, stimulation of β1-adrenergic receptors produces cAMP production, but surprisingly, β1-adrenergic receptor activation of adenylyl cyclase was not functionally linked to rapid CREB phosphorylation. These findings demonstrate that activation of β1-adrenergic receptors on striatal neurons can stimulate two distinct signaling pathways. These adrenergic actions can produce long-term changes in gene expression, as well as rapidly modulate cellular physiology. By elucidating the mechanisms by which norepinephrine and β1-adrenergic receptor activation affects striatal physiology, we provide the means to more fully understand the role of monoamines in modulating striatal function, specifically how norepinephrine and β1-adrenergic receptors may affect striatal physiology. PMID:21143600

  6. Kinase cascades and ligand-directed signaling at the kappa opioid receptor.

    PubMed

    Bruchas, Michael R; Chavkin, Charles

    2010-06-01

    The dynorphin/kappa opioid receptor (KOR) system has been implicated as a critical component of the stress response. Stress-induced activation of dynorphin-KOR is well known to produce analgesia, and more recently, it has been implicated as a mediator of stress-induced responses including anxiety, depression, and reinstatement of drug seeking. Drugs selectively targeting specific KOR signaling pathways may prove potentially useful as therapeutic treatments for mood and addiction disorders. KOR is a member of the seven transmembrane spanning (7TM) G-protein coupled receptor (GPCR) superfamily. KOR activation of pertussis toxin-sensitive G proteins leads to Galphai/o inhibition of adenylyl cyclase production of cAMP and releases Gbetagamma, which modulates the conductances of Ca(+2) and K(+) channels. In addition, KOR agonists activate kinase cascades including G-protein coupled Receptor Kinases (GRK) and members of the mitogen-activated protein kinase (MAPK) family: ERK1/2, p38 and JNK. Recent pharmacological data suggests that GPCRs exist as dynamic, multi-conformational protein complexes that can be directed by specific ligands towards distinct signaling pathways. Ligand-induced conformations of KOR that evoke beta-arrestin-dependent p38 MAPK activation result in aversion; whereas ligand-induced conformations that activate JNK without activating arrestin produce long-lasting inactivation of KOR signaling. In this review, we discuss the current status of KOR signal transduction research and the data that support two novel hypotheses: (1) KOR selective partial agonists that do not efficiently activate p38 MAPK may be useful analgesics without producing the dysphoric or hallucinogenic effects of selective, highly efficacious KOR agonists and (2) KOR antagonists that do not activate JNK may be effective short-acting drugs that may promote stress-resilience.

  7. Prostaglandin E2 Inhibits Histamine-Evoked Ca2+ Release in Human Aortic Smooth Muscle Cells through Hyperactive cAMP Signaling Junctions and Protein Kinase A

    PubMed Central

    Taylor, Emily J. A.; Pantazaka, Evangelia; Shelley, Kathryn L.

    2017-01-01

    In human aortic smooth muscle cells, prostaglandin E2 (PGE2) stimulates adenylyl cyclase (AC) and attenuates the increase in intracellular free Ca2+ concentration evoked by activation of histamine H1 receptors. The mechanisms are not resolved. We show that cAMP mediates inhibition of histamine-evoked Ca2+ signals by PGE2. Exchange proteins activated by cAMP were not required, but the effects were attenuated by inhibition of cAMP-dependent protein kinase (PKA). PGE2 had no effect on the Ca2+ signals evoked by protease-activated receptors, heterologously expressed muscarinic M3 receptors, or by direct activation of inositol 1,4,5-trisphosphate (IP3) receptors by photolysis of caged IP3. The rate of Ca2+ removal from the cytosol was unaffected by PGE2, but PGE2 attenuated histamine-evoked IP3 accumulation. Substantial inhibition of AC had no effect on the concentration-dependent inhibition of Ca2+ signals by PGE2 or butaprost (to activate EP2 receptors selectively), but it modestly attenuated responses to EP4 receptors, activation of which generated less cAMP than EP2 receptors. We conclude that inhibition of histamine-evoked Ca2+ signals by PGE2 occurs through “hyperactive signaling junctions,” wherein cAMP is locally delivered to PKA at supersaturating concentrations to cause uncoupling of H1 receptors from phospholipase C. This sequence allows digital signaling from PGE2 receptors, through cAMP and PKA, to histamine-evoked Ca2+ signals. PMID:28877931

  8. Characterization of nitrosoalkane binding and activation of soluble guanylate cyclase.

    PubMed

    Derbyshire, Emily R; Tran, Rosalie; Mathies, Richard A; Marletta, Michael A

    2005-12-13

    Soluble guanylate cyclase (sGC) is the primary receptor for the signaling agent nitric oxide (NO). Electronic absorption and resonance Raman spectroscopy were used to show that nitrosoalkanes bind to the heme of sGC to form six-coordinate, low-spin complexes. In the sGC-nitrosopentane complex, a band assigned to an Fe-N stretching vibration is observed at 543 cm(-)(1) which is similar to values reported for other six-coordinate NO-bound hemoproteins. Nitrosoalkanes activate sGC 2-6-fold and synergize with YC-1, a synthetic benzylindazole derivative, to activate the enzyme 11-47-fold. In addition, the observed off-rates of nitrosoalkanes from sGC were found to be dependent on the alkyl chain length. A linear correlation was found between the observed off-rates and the alkyl chain length which suggests that the sGC heme has a large hydrophobic distal ligand-binding pocket. Together, these data show that nitrosoalkanes are a novel class of heme-based sGC activators and suggest that heme ligation is a general requirement for YC-1 synergism.

  9. Expansion of signal transduction by G proteins The second 15 years or so: From 3 to 16 α subunits plus βγ dimers

    PubMed Central

    Birnbaumer, Lutz

    2007-01-01

    The first 15 years, or so, brought the realization that there existed a G protein coupled signal transduction mechanism by which hormone receptors regulate adenylyl cyclases and the light receptor rhodopsin activates visual phosphodiesterase. Three G proteins, Gs, Gi and transducin (T) had been characterized as αβγ heterotrimers, and Gsα-GTP and Tα-GTP had been identified as the sigaling arms of Gs and T. These discoveries were made using classical biochemical approaches, and culminated in the purification of these G proteins. The second 15 years, or so, are the subject of the present review. This time coincided with the advent of powerful recombinant DNA techniques. Combined with the classical approaches, the field expanded the repertoire of G proteins from 3 to 16, discovered the superfamily of seven transmembrane G protein coupled receptors (GPCRs) – which is not addressed in this article – and uncovered an amazing repertoire of effector functions regulated not only by αGTP complexes but also by βγ dimers. Emphasis is placed in presenting how the field developed with the hope of conveying why many of the new findings were made. PMID:17258171

  10. Multiple Transduction Pathways Mediate Thyrotropin Receptor Signaling in Preosteoblast-Like Cells

    PubMed Central

    Boutin, Alisa; Neumann, Susanne

    2016-01-01

    It has been shown that the TSH receptor (TSHR) couples to a number of different signaling pathways, although the Gs-cAMP pathway has been considered primary. Here, we measured the effects of TSH on bone marker mRNA and protein expression in preosteoblast-like U2OS cells stably expressing TSHRs. We determined which signaling cascades are involved in the regulation of IL-11, osteopontin (OPN), and alkaline phosphatase (ALPL). We demonstrated that TSH-induced up-regulation of IL-11 is primarily mediated via the Gs pathway as IL-11 was up-regulated by forskolin (FSK), an adenylyl cyclase activator, and inhibited by protein kinase A inhibitor H-89 and by silencing of Gαs by small interfering RNA. OPN levels were not affected by FSK, but its up-regulation was inhibited by TSHR/Gi-uncoupling by pertussis toxin. Pertussis toxin decreased p38 MAPK kinase phosphorylation, and a p38 inhibitor and small interfering RNA knockdown of p38α inhibited OPN induction by TSH. Up-regulation of ALPL expression required high doses of TSH (EC50 = 395nM), whereas low doses (EC50 = 19nM) were inhibitory. FSK-stimulated cAMP production decreased basal ALPL expression, whereas protein kinase A inhibition by H-89 and silencing of Gαs increased basal levels of ALPL. Knockdown of Gαq/11 and a protein kinase C inhibitor decreased TSH-stimulated up-regulation of ALPL, whereas a protein kinase C activator increased ALPL levels. A MAPK inhibitor and silencing of ERK1/2 inhibited TSH-stimulated ALPL expression. We conclude that TSH regulates expression of different bone markers via distinct signaling pathways. PMID:26950201

  11. Molecular, pharmacological, and signaling properties of octopamine receptors from honeybee (Apis mellifera) brain.

    PubMed

    Balfanz, Sabine; Jordan, Nadine; Langenstück, Teresa; Breuer, Johanna; Bergmeier, Vera; Baumann, Arnd

    2014-04-01

    G protein-coupled receptors are important regulators of cellular signaling processes. Within the large family of rhodopsin-like receptors, those binding to biogenic amines form a discrete subgroup. Activation of biogenic amine receptors leads to transient changes of intracellular Ca²⁺-([Ca²⁺](i)) or 3',5'-cyclic adenosine monophosphate ([cAMP](i)) concentrations. Both second messengers modulate cellular signaling processes and thereby contribute to long-lasting behavioral effects in an organism. In vivo pharmacology has helped to reveal the functional effects of different biogenic amines in honeybees. The phenolamine octopamine is an important modulator of behavior. Binding of octopamine to its receptors causes elevation of [Ca²⁺](i) or [cAMP](i). To date, only one honeybee octopamine receptor that induces Ca²⁺ signals has been molecularly and pharmacologically characterized. Here, we examined the pharmacological properties of four additional honeybee octopamine receptors. When heterologously expressed, all receptors induced cAMP production after binding to octopamine with EC₅₀(s) in the nanomolar range. Receptor activity was most efficiently blocked by mianserin, a substance with antidepressant activity in vertebrates. The rank order of inhibitory potency for potential receptor antagonists was very similar on all four honeybee receptors with mianserin > cyproheptadine > metoclopramide > chlorpromazine > phentolamine. The subroot of octopamine receptors activating adenylyl cyclases is the largest that has so far been characterized in arthropods, and it should now be possible to unravel the contribution of individual receptors to the physiology and behavior of honeybees. © 2013 International Society for Neurochemistry.

  12. Multiple Facets of cAMP Signalling and Physiological Impact: cAMP Compartmentalization in the Lung

    PubMed Central

    Oldenburger, Anouk; Maarsingh, Harm; Schmidt, Martina

    2012-01-01

    Therapies involving elevation of the endogenous suppressor cyclic AMP (cAMP) are currently used in the treatment of several chronic inflammatory disorders, including chronic obstructive pulmonary disease (COPD). Characteristics of COPD are airway obstruction, airway inflammation and airway remodelling, processes encompassed by increased airway smooth muscle mass, epithelial changes, goblet cell and submucosal gland hyperplasia. In addition to inflammatory cells, airway smooth muscle cells and (myo)fibroblasts, epithelial cells underpin a variety of key responses in the airways such as inflammatory cytokine release, airway remodelling, mucus hypersecretion and airway barrier function. Cigarette smoke, being next to environmental pollution the main cause of COPD, is believed to cause epithelial hyperpermeability by disrupting the barrier function. Here we will focus on the most recent progress on compartmentalized signalling by cAMP. In addition to G protein-coupled receptors, adenylyl cyclases, cAMP-specific phospho-diesterases (PDEs) maintain compartmentalized cAMP signalling. Intriguingly, spatially discrete cAMP-sensing signalling complexes seem also to involve distinct members of the A-kinase anchoring (AKAP) superfamily and IQ motif containing GTPase activating protein (IQGAPs). In this review, we will highlight the interaction between cAMP and the epithelial barrier to retain proper lung function and to alleviate COPD symptoms and focus on the possible molecular mechanisms involved in this process. Future studies should include the development of cAMP-sensing multiprotein complex specific disruptors and/or stabilizers to orchestrate cellular functions. Compartmentalized cAMP signalling regulates important cellular processes in the lung and may serve as a therapeutic target. PMID:24281338

  13. Structure-guided design and functional characterization of an artificial red light-regulated guanylate/adenylate cyclase for optogenetic applications.

    PubMed

    Etzl, Stefan; Lindner, Robert; Nelson, Matthew D; Winkler, Andreas

    2018-06-08

    Genetically targeting biological systems to control cellular processes with light is the concept of optogenetics. Despite impressive developments in this field, underlying molecular mechanisms of signal transduction of the employed photoreceptor modules are frequently not sufficiently understood to rationally design new optogenetic tools. Here, we investigate the requirements for functional coupling of red light-sensing phytochromes with non-natural enzymatic effectors by creating a series of constructs featuring the Deinococcus radiodurans bacteriophytochrome linked to a Synechocystis guanylate/adenylate cyclase. Incorporating characteristic structural elements important for cyclase regulation in our designs, we identified several red light-regulated fusions with promising properties. We provide details of one light-activated construct with low dark-state activity and high dynamic range that outperforms previous optogenetic tools in vitro and expands our in vivo toolkit, as demonstrated by manipulation of Caenorhabditis elegans locomotor activity. The full-length crystal structure of this phytochrome-linked cyclase revealed molecular details of photoreceptor-effector coupling, highlighting the importance of the regulatory cyclase element. Analysis of conformational dynamics by hydrogen-deuterium exchange in different functional states enriched our understanding of phytochrome signaling and signal integration by effectors. We found that light-induced conformational changes in the phytochrome destabilize the coiled-coil sensor-effector linker, which releases the cyclase regulatory element from an inhibited conformation, increasing cyclase activity of this artificial system. Future designs of optogenetic functionalities may benefit from our work, indicating that rational considerations for the effector improve the rate of success of initial designs to obtain optogenetic tools with superior properties. © 2018 Etzl et al.

  14. The Yersinia pestis Rcs phosphorelay inhibits biofilm formation by repressing transcription of the diguanylate cyclase gene hmsT.

    PubMed

    Sun, Yi-Cheng; Guo, Xiao-Peng; Hinnebusch, B Joseph; Darby, Creg

    2012-04-01

    Yersinia pestis, which causes bubonic plague, forms biofilms in fleas, its insect vectors, as a means to enhance transmission. Biofilm development is positively regulated by hmsT, encoding a diguanylate cyclase that synthesizes the bacterial second messenger cyclic-di-GMP. Biofilm development is negatively regulated by the Rcs phosphorelay signal transduction system. In this study, we show that Rcs-negative regulation is accomplished by repressing transcription of hmsT.

  15. Adenylate Cyclases of Trypanosoma brucei, Environmental Sensors and Controllers of Host Innate Immune Response.

    PubMed

    Salmon, Didier

    2018-04-25

    Trypanosoma brucei , etiological agent of Sleeping Sickness in Africa, is the prototype of African trypanosomes, protozoan extracellular flagellate parasites transmitted by saliva ( Salivaria ). In these parasites the molecular controls of the cell cycle and environmental sensing are elaborate and concentrated at the flagellum. Genomic analyses suggest that these parasites appear to differ considerably from the host in signaling mechanisms, with the exception of receptor-type adenylate cyclases (AC) that are topologically similar to receptor-type guanylate cyclase (GC) of higher eukaryotes but control a new class of cAMP targets of unknown function, the cAMP response proteins (CARPs), rather than the classical protein kinase A cAMP effector (PKA). T. brucei possesses a large polymorphic family of ACs, mainly associated with the flagellar membrane, and these are involved in inhibition of the innate immune response of the host prior to the massive release of immunomodulatory factors at the first peak of parasitemia. Recent evidence suggests that in T. brucei several insect-specific AC isoforms are involved in social motility, whereas only a few AC isoforms are involved in cytokinesis control of bloodstream forms, attesting that a complex signaling pathway is required for environmental sensing. In this review, after a general update on cAMP signaling pathway and the multiple roles of cAMP, I summarize the existing knowledge of the mechanisms by which pathogenic microorganisms modulate cAMP levels to escape immune defense.

  16. Regulation of P450-mediated permethrin resistance in Culex quinquefasciatus by the GPCR/Gαs/AC/cAMP/PKA signaling cascade.

    PubMed

    Li, Ting; Liu, Nannan

    2017-12-01

    This study explores the role of G-protein-coupled receptor-intracellular signaling in the development of P450-mediated insecticide resistance in mosquitoes, Culex quinquefasciatus , focusing on the essential function of the GPCRs and their downstream effectors of Gs alpha subunit protein (Gαs) and adenylyl cyclase (ACs) in P450-mediated insecticide resistance of Culex mosquitoes. Our RNAi-mediated functional study showed that knockdown of Gαs caused the decreased expression of the downstream effectors of ACs and PKAs in the GPCR signaling pathway and resistance P450 genes, whereas knockdown of ACs decreased the expression of PKAs and resistance P450 genes. Knockdown of either Gαs or ACs resulted in an increased susceptibility of mosquitoes to permethrin. These results add significantly to our understanding of the molecular basis of resistance P450 gene regulation through GPCR/Gαs/AC/cAMP-PKA signaling pathways in the insecticide resistance of mosquitoes. The temporal and spatial dynamic analyses of GPCRs, Gαs, ACs, PKAs, and P450s in two insecticide resistant mosquito strains revealed that all the GPCR signaling pathway components tested, namely GPCRs, Gαs, ACs and PKAs, were most highly expressed in the brain for both resistant strains, suggesting the role played by these genes in signaling transduction and regulation. The resistance P450 genes were mainly expressed in the brain, midgut and malpighian tubules (MTs), suggesting their critical function in the central nervous system and importance for detoxification. The temporal dynamics analysis for the gene expression showed a diverse expression profile during mosquito development, indicating their initially functional importance in response to exposure to insecticides during their life stages.

  17. Dynamics of β-adrenergic/cAMP signaling and morphological changes in cultured astrocytes.

    PubMed

    Vardjan, Nina; Kreft, Marko; Zorec, Robert

    2014-04-01

    The morphology of astrocytes, likely regulated by cAMP, determines the structural association between astrocytes and the synapse, consequently modulating synaptic function. β-Adrenergic receptors (β-AR), which increase cytosolic cAMP concentration ([cAMP]i ), may affect cell morphology. However, the real-time dynamics of β-AR-mediated cAMP signaling in single live astrocytes and its effect on cell morphology have not been studied. We used the fluorescence resonance energy transfer (FRET)-based cAMP biosensor Epac1-camps to study time-dependent changes in [cAMP]i ; morphological changes in primary rat astrocytes were monitored by real-time confocal microscopy. Stimulation of β-AR by adrenaline, noradrenaline, and isoprenaline, a specific agonist of β-AR, rapidly increased [cAMP]i (∼15 s). The FRET signal response, mediated via β-AR, was faster than in the presence of forskolin (twofold) and dibutyryl-cAMP (>35-fold), which directly activate adenylyl cyclase and Epac1-camps, respectively, likely due to slow entry of these agents into the cytosol. Oscillations in [cAMP]i have not been recorded, indicating that cAMP-dependent processes operate in a slow time domain. Most Epac1-camps expressing astrocytes revealed a morphological change upon β-AR activation and attained a stellate morphology within 1 h. The morphological changes exhibited a bell-shaped dependency on [cAMP]i . The 5-10% decrease in cell cross-sectional area and the 30-50% increase in cell perimeter are likely due to withdrawal of the cytoplasm to the perinuclear region and the appearance of protrusions on the surface of astrocytes. Because astrocyte processes ensheath neurons, β-AR/cAMP-mediated morphological changes can modify the geometry of the extracellular space, affecting synaptic, neuronal, and astrocyte functions in health and disease. Copyright © 2014 Wiley Periodicals, Inc.

  18. DMT efficiently inhibits hepatic gluconeogenesis by regulating the Gαq signaling pathway.

    PubMed

    Zhou, Ting-Ting; Ma, Fei; Shi, Xiao-Fan; Xu, Xin; Du, Te; Guo, Xiao-Dan; Wang, Gai-Hong; Yu, Liang; Rukachaisirikul, Vatcharin; Hu, Li-Hong; Chen, Jing; Shen, Xu

    2017-08-01

    Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease with complicated pathogenesis and targeting gluconeogenesis inhibition is a promising strategy for anti-diabetic drug discovery. G protein-coupled receptors (GPCRs) are classified as distinct families by heterotrimeric G proteins, primarily including Gαs, Gαi and Gαq. Gαs-coupled GPCRs function potently in the regulation of hepatic gluconeogenesis by activating cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway and Gαi-coupled GPCRs exhibit inhibitory effect on adenylyl cyclase and reduce intracellular cAMP level. However, little is known about the regulation of Gαq-coupled GPCRs in hepatic gluconeogenesis. Here, small-molecule 2-(2,4-dimethoxy-3-methylphenyl)-7-(thiophen-2-yl)-9-(trifluoromethyl)-2,3-dihydropyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4( 1H )-one (DMT) was determined to suppress hepatic glucose production and reduce mRNA levels of gluconeogenic genes. Treatment of DMT in db/db mice decreased fasting blood glucose and hemoglobin A1C (HbA1c) levels, while improved glucose tolerance and pyruvate tolerance. Mechanism study demonstrated that DMT-inhibited gluconeogenesis by regulating the Gαq/phospholipase C (PLC)/inositol-1,4,5-triphosphate receptor (IP3R)-mediated calcium (Ca 2+ )/calmodulin (CaM)/phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT)/forkhead box protein O1 (FOXO1) signaling pathway. To our knowledge, DMT might be the first reported small molecule able to suppress hepatic gluconeogenesis by regulating Gαq signaling, and our current work has also highlighted the potential of DMT in the treatment of T2DM. © 2017 Society for Endocrinology.

  19. Signaling mechanisms mediating muscarinic enhancement of GABAergic synaptic transmission in the spinal cord.

    PubMed

    Zhang, H-M; Chen, S-R; Cai, Y-Q; Richardson, T E; Driver, L C; Lopez-Berestein, G; Pan, H-L

    2009-02-18

    Activation of muscarinic acetylcholine receptors (mAChRs) inhibits spinal nociceptive transmission by potentiation of GABAergic tone through M(2), M(3), and M(4) subtypes. To study the signaling mechanisms involved in this unique mAChR action, GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) of lamina II neurons were recorded using whole-cell patch clamp techniques in rat spinal cord slices. The mAChR agonist oxotremorine-M caused a profound increase in the frequency of GABAergic sIPSCs, which was abolished in the Ca(2+)-free solution. Inhibition of voltage-gated Ca(2+) channels with Cd(2+) and Ni(2+) largely reduced the effect of oxotremorine-M on sIPSCs. Blocking nonselective cation channels (NSCCs) with SKF96365 or 2-APB also largely attenuated the effect of oxotremorine-M. However, the KCNQ channel blocker XE991 and the adenylyl cyclase inhibitor MDL12330A had no significant effect on oxotremorine-M-induced increases in sIPSCs. Furthermore, the phosphoinositide-3-kinase (PI3K) inhibitor wortmannin or LY294002 significantly reduced the potentiating effect of oxotremorine-M on sIPSCs. In the spinal cord in which the M(3) subtype was specifically knocked down by intrathecal small interfering RNA (siRNA) treatment, SKF96365 and wortmannin still significantly attenuated the effect of oxotremorine-M. In contrast, SKF96365 and wortmannin both failed to alter the effect of oxotremorine-M on sIPSCs when the M(2)/M(4) mAChRs were blocked. Therefore, our study provides new evidence that activation of mAChRs increases synaptic GABA release through Ca(2+) influx and voltage-gated Ca(2+) channels. The PI3K-NSCC signaling cascade is primarily involved in the excitation of GABAergic interneurons by the M(2)/M(4) mAChRs in the spinal dorsal horn.

  20. Activation of cAMP-dependent signaling pathway induces mouse organic anion transporting polypeptide 2 expression.

    PubMed

    Chen, Chuan; Cheng, Xingguo; Dieter, Matthew Z; Tanaka, Yuji; Klaassen, Curtis D

    2007-04-01

    Rodent Oatp2 is a hepatic uptake transporter for such compounds as cardiac glycosides. In the present study, we found that fasting resulted in a 2-fold induction of Oatp2 expression in liver of mice. Because the cAMP-protein kinase A (PKA) signaling pathway is activated during fasting, the role of this pathway in Oatp2 induction during fasting was examined. In Hepa-1c1c7 cells, adenylyl cyclase activator forskolin as well as two cellular membrane-permeable cAMP analogs, dibutyryl cAMP and 8-bromo-cAMP, induced Oatp2 mRNA expression in a time- and dose-dependent manner. These three chemicals induced reporter gene activity in cells transfected with a luciferase reporter gene construct containing a 7.6-kilobase (kb) 5'-flanking region of mouse Oatp2. Transient transfection of cells with 5'-deletion constructs derived from the 7.6-kb Oatp2 promoter reporter gene construct, as well as 7.6-kb constructs in which a consensus cAMP response element (CRE) half-site CGTCA (-1808/-1804 bp) was mutated or deleted, confirms that this CRE site was required for the induction of luciferase activity by forskolin. Luciferase activity driven by the Oatp2 promoter containing this CRE site was induced in cells cotransfected with a plasmid encoding the protein kinase A catalytic subunit. Cotransfection of cells with a plasmid encoding the dominant-negative CRE binding protein (CREB) completely abolished the inducibility of the reporter gene activity by forskolin. In conclusion, induction of Oatp2 expression in liver of fasted mice may be caused by activation of the cAMP-dependent signaling pathway, with the CRE site (-1808/-1804) and CREB being the cis- and trans-acting factors mediating the induction, respectively.

  1. [Adenylate cyclase from rabbit heart: substrate binding site].

    PubMed

    Perfil'eva, E A; Khropov, Iu V; Khachatrian, L; Bulargina, T V; Baranova, L A

    1981-08-01

    The effects of 17 ATP analogs on the solubilized rabbit heart adenylate cyclase were studied. The triphosphate chain, position 8 of the adenine base and the ribose residue of the ATP molecule were modified. Despite the presence of the alkylating groups in two former types of the analogs tested, no covalent blocking of the active site of the enzyme was observed. Most of the compounds appeared to be competitive reversible inhibitors. The kinetic data confirmed the importance of the triphosphate chain for substrate binding in the active site of adenylate cyclase. (Formula: See Text) The inhibitors with different substituents in position 8 of the adenine base had a low affinity for the enzyme. The possible orientation of the triphosphate chain and the advantages of anti-conformation of the ATP molecule for their binding in the active site of adenylate cyclase are discussed.

  2. TSH Receptor Signaling Abrogation by a Novel Small Molecule

    PubMed Central

    Latif, Rauf; Realubit, Ronald B.; Karan, Charles; Mezei, Mihaly; Davies, Terry F.

    2016-01-01

    Pathological activation of the thyroid-stimulating hormone receptor (TSHR) is caused by thyroid-stimulating antibodies in patients with Graves’ disease (GD) or by somatic and rare genomic mutations that enhance constitutive activation of the receptor influencing both G protein and non-G protein signaling. Potential selective small molecule antagonists represent novel therapeutic compounds for abrogation of such abnormal TSHR signaling. In this study, we describe the identification and in vitro characterization of a novel small molecule antagonist by high-throughput screening (HTS). The identification of the TSHR antagonist was performed using a transcription-based TSH-inhibition bioassay. TSHR-expressing CHO cells, which also expressed a luciferase-tagged CRE response element, were optimized using bovine TSH as the activator, in a 384 well plate format, which had a Z score of 0.3–0.6. Using this HTS assay, we screened a diverse library of ~80,000 compounds at a final concentration of 16.7 μM. The selection criteria for a positive hit were based on a mean signal threshold of ≥50% inhibition of control TSH stimulation. The screening resulted in 450 positive hits giving a hit ratio of 0.56%. A secondary confirmation screen against TSH and forskolin – a post receptor activator of adenylyl cyclase – confirmed one TSHR-specific candidate antagonist molecule (named VA-K-14). This lead molecule had an IC50 of 12.3 μM and a unique chemical structure. A parallel analysis for cell viability indicated that the lead inhibitor was non-cytotoxic at its effective concentrations. In silico docking studies performed using a TSHR transmembrane model showed the hydrophobic contact locations and the possible mode of inhibition of TSHR signaling. Furthermore, this molecule was capable of inhibiting TSHR stimulation by GD patient sera and monoclonal-stimulating TSHR antibodies. In conclusion, we report the identification of a novel small molecule TSHR inhibitor, which has

  3. Exchange protein activated by cyclic AMP (Epac)-mediated induction of suppressor of cytokine signaling 3 (SOCS-3) in vascular endothelial cells.

    PubMed

    Sands, William A; Woolson, Hayley D; Milne, Gillian R; Rutherford, Claire; Palmer, Timothy M

    2006-09-01

    Here, we demonstrate that elevation of intracellular cyclic AMP (cAMP) in vascular endothelial cells (ECs) by either a direct activator of adenylyl cyclase or endogenous cAMP-mobilizing G protein-coupled receptors inhibited the tyrosine phosphorylation of STAT proteins by an interleukin 6 (IL-6) receptor trans-signaling complex (soluble IL-6Ralpha/IL-6). This was associated with the induction of suppressor of cytokine signaling 3 (SOCS-3), a bona fide inhibitor in vivo of gp130, the signal-transducing component of the IL-6 receptor complex. Attenuation of SOCS-3 induction in either ECs or SOCS-3-null murine embryonic fibroblasts abolished the inhibitory effect of cAMP, whereas inhibition of SHP-2, another negative regulator of gp130, was without effect. Interestingly, the inhibition of STAT phosphorylation and SOCS-3 induction did not require cAMP-dependent protein kinase activity but could be recapitulated upon selective activation of the alternative cAMP sensor Epac, a guanine nucleotide exchange factor for Rap1. Consistent with this hypothesis, small interfering RNA-mediated knockdown of Epac1 was sufficient to attenuate both cAMP-mediated SOCS-3 induction and inhibition of STAT phosphorylation, suggesting that Epac activation is both necessary and sufficient to observe these effects. Together, these data argue for the existence of a novel cAMP/Epac/Rap1/SOCS-3 pathway for limiting IL-6 receptor signaling in ECs and illuminate a new mechanism by which cAMP may mediate its potent anti-inflammatory effects.

  4. Regulation of mGlu4 metabotropic glutamate receptor signaling by type-2 G-protein coupled receptor kinase (GRK2).

    PubMed

    Iacovelli, L; Capobianco, L; Iula, M; Di Giorgi Gerevini, V; Picascia, A; Blahos, J; Melchiorri, D; Nicoletti, F; De Blasi, A

    2004-05-01

    We examined the role of G-protein coupled receptor kinase-2 (GRK2) in the homologous desensitization of mGlu4 metabotropic glutamate receptors transiently expressed in human embryonic kidney (HEK) 293 cells. Receptor activation with the agonist l-2-amino-4-phosphonobutanoate (l-AP4) stimulated at least two distinct signaling pathways: inhibition of cAMP formation and activation of the mitogen-activated protein kinase (MAPK) pathway [assessed by Western blot analysis of phosphorylated extracellular signal-regulated kinase (ERK) 1 and 2]. Activation of both pathways was attenuated by pertussis toxin. Overexpression of GRK2 (but not GRK4) largely attenuated the stimulation of the MAPK pathway by l-AP4, whereas it slightly potentiated the inhibition of FSK-stimulated cAMP formation. Transfection with a kinase-dead mutant of GRK2 (GRK2-K220R) or with the C-terminal fragment of GRK2 also reduced the mGlu4-mediated stimulation of MAPK, suggesting that GRK2 binds to the Gbetagamma subunits to inhibit signal propagation toward the MAPK pathway. This was confirmed by the evidence that GRK2 coimmunoprecipitated with Gbetagamma subunits in an agonist-dependent manner. Finally, neither GRK2 nor its kinase-dead mutant had any effect on agonist-induced mGlu4 receptor internalization in HEK293 cells transiently transfected with GFP-tagged receptors. Agonist-dependent internalization was instead abolished by a negative-dominant mutant of dynamin, which also reduced the stimulation of MAPK pathway by l-AP4. We speculate that GRK2 acts as a "switch molecule" by inhibiting the mGlu4 receptor-mediated stimulation of MAPK and therefore directing the signal propagation toward the inhibition of adenylyl cyclase.

  5. Cyclic AMP-Elevating Capacity of Adenylate Cyclase Toxin-Hemolysin Is Sufficient for Lung Infection but Not for Full Virulence of Bordetella pertussis

    PubMed Central

    Skopova, Karolina; Tomalova, Barbora; Kanchev, Ivan; Rossmann, Pavel; Svedova, Martina; Adkins, Irena; Bibova, Ilona; Tomala, Jakub; Masin, Jiri; Guiso, Nicole; Osicka, Radim; Sedlacek, Radislav; Kovar, Marek

    2017-01-01

    ABSTRACT The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, αMβ2 integrin, or CD11b/CD18). CyaA delivers into cells an N-terminal adenylyl cyclase (AC) enzyme domain that is activated by cytosolic calmodulin and catalyzes unregulated conversion of cellular ATP into cyclic AMP (cAMP), a key second messenger subverting bactericidal activities of phagocytes. In parallel, the hemolysin (Hly) moiety of CyaA forms cation-selective hemolytic pores that permeabilize target cell membranes. We constructed the first B. pertussis mutant secreting a CyaA toxin having an intact capacity to deliver the AC enzyme into CD11b-expressing (CD11b+) host phagocytes but impaired in formation of cell-permeabilizing pores and defective in cAMP elevation in CD11b− cells. The nonhemolytic AC+ Hly− bacteria inhibited the antigen-presenting capacities of coincubated mouse dendritic cells in vitro and skewed their Toll-like receptor (TLR)-triggered maturation toward a tolerogenic phenotype. The AC+ Hly− mutant also infected mouse lungs as efficiently as the parental AC+ Hly+ strain. Hence, elevation of cAMP in CD11b− cells and/or the pore-forming capacity of CyaA were not required for infection of mouse airways. The latter activities were, however, involved in bacterial penetration across the epithelial layer, enhanced neutrophil influx into lung parenchyma during sublethal infections, and the exacerbated lung pathology and lethality of B. pertussis infections at higher inoculation doses (>107 CFU/mouse). The pore-forming activity of CyaA further synergized with the cAMP-elevating activity in downregulation of major histocompatibility complex class II (MHC-II) molecules on infiltrating myeloid cells, likely contributing to immune subversion of host defenses by the whooping cough agent. PMID:28396322

  6. Adenylyl cylases 1 and 8 mediate select striatal-dependent behaviors and sensitivity to ethanol stimulation in the adolescent period following acute neonatal ethanol exposure.

    PubMed

    Susick, Laura L; Lowing, Jennifer L; Bosse, Kelly E; Hildebrandt, Clara C; Chrumka, Alexandria C; Conti, Alana C

    2014-08-01

    Neonatal alcohol exposure in rodents causes dramatic neurodegenerative effects throughout the developing nervous system, particularly in the striatum, acutely after exposure. These acute neurodegenerative effects are augmented in mice lacking adenylyl cyclases 1 and 8 (AC1/8) as neonatal mice with a genetic deletion of both AC isoforms (DKO) have increased vulnerability to ethanol-induced striatal neurotoxicity compared to wild type (WT) controls. While neonatal ethanol exposure is known to negatively impact cognitive behaviors, such as executive functioning and working memory in adolescent and adult animals, the threshold of ethanol exposure required to impinge upon developmental behaviors in mice has not been extensively examined. Therefore, the purpose of this study was to determine the behavioral effects of neonatal ethanol exposure using various striatal-dependent developmental benchmarks and to assess the impact of AC1/8 deletion on this developmental progression. WT and DKO mice were treated with 2.5 g/kg ethanol or saline on postnatal day (P)6 and later subjected to the wire suspension, negative geotaxis, postural reflex, grid hang, tail suspension and accelerating rotarod tests at various time points. At P30, mice were evaluated for their hypnotic responses to 4.0 g/kg ethanol by using the loss of righting reflex assay and ethanol-induced stimulation of locomotor activity after 2.0 g/kg ethanol. Ethanol exposure significantly impaired DKO performance in the negative geotaxis test while genetic deletion of AC1/8 alone increased grid hang time and decreased immobility time in the tail suspension test with a concomitant increase in hindlimb clasping behavior. Locomotor stimulation was significantly increased in animals that received ethanol as neonates, peaking significantly in ethanol-treated DKO mice compared to ethanol-treated WT controls, while sedation duration following high-dose ethanol challenge was unaffected. These data indicate that the

  7. Diphtheria toxin can simultaneously bind to its receptor and adenylyl-(3',5')-uridine 3'-monophosphate

    SciTech Connect

    Barbieri, J.T.; Collins, C.M.; Collier, R.J.

    1986-10-21

    Diphtheria toxin (DT) that was bound to receptors on BS-C-1 cells was able to bind approximately 1 molar equiv of adenylyl-(3',5')-uridine 3'-monophosphate (ApUp). In contrast, receptor-bound CRM197, a mutant form of toxin with greatly diminished affinity for dinucleotides, did not bind ApUp. Affinity of the dinucleotide for receptor-bound toxin differed from that for free toxin by less than an order of magnitude. These results indicate that the receptor site and the ApUp site on the toxin do not significantly overlap. BS-C-1 cells were incubated with or without /sup 125/I-DT or CRM 197. They were then incubated with (/sup 32/P)ApUp, andmore » assayed.« less

  8. General base-general acid catalysis by terpenoid cyclases.

    PubMed

    Pemberton, Travis A; Christianson, David W

    2016-07-01

    Terpenoid cyclases catalyze the most complex reactions in biology, in that more than half of the substrate carbon atoms often undergo changes in bonding during the course of a multistep cyclization cascade that proceeds through multiple carbocation intermediates. Many cyclization mechanisms require stereospecific deprotonation and reprotonation steps, and most cyclization cascades are terminated by deprotonation to yield an olefin product. The first bacterial terpenoid cyclase to yield a crystal structure was pentalenene synthase from Streptomyces exfoliatus UC5319. This cyclase generates the hydrocarbon precursor of the pentalenolactone family of antibiotics. The structures of pentalenene synthase and other terpenoid cyclases reveal predominantly nonpolar active sites typically lacking amino acid side chains capable of serving general base-general acid functions. What chemical species, then, enables the Brønsted acid-base chemistry required in the catalytic mechanisms of these enzymes? The most likely candidate for such general base-general acid chemistry is the co-product inorganic pyrophosphate. Here, we briefly review biological and nonbiological systems in which phosphate and its derivatives serve general base and general acid functions in catalysis. These examples highlight the fact that the Brønsted acid-base activities of phosphate derivatives are comparable to the Brønsted acid-base activities of amino acid side chains.

  9. Deep Conservation of Genes Required for Both Drosophila melanogaster and Caenorhabditis elegans Sleep Includes a Role for Dopaminergic Signaling

    PubMed Central

    Singh, Komudi; Ju, Jennifer Y.; Walsh, Melissa B.; DiIorio, Michael A.; Hart, Anne C.

    2014-01-01

    Objectives: Cross-species conservation of sleep-like behaviors predicts the presence of conserved molecular mechanisms underlying sleep. However, limited experimental evidence of conservation exists. Here, this prediction is tested directly. Measurements and Results: During lethargus, Caenorhabditis elegans spontaneously sleep in short bouts that are interspersed with bouts of spontaneous locomotion. We identified 26 genes required for Drosophila melanogaster sleep. Twenty orthologous C. elegans genes were selected based on similarity. Their effect on C. elegans sleep and arousal during the last larval lethargus was assessed. The 20 most similar genes altered both the quantity of sleep and arousal thresholds. In 18 cases, the direction of change was concordant with Drosophila studies published previously. Additionally, we delineated a conserved genetic pathway by which dopamine regulates sleep and arousal. In C. elegans neurons, G-alpha S, adenylyl cyclase, and protein kinase A act downstream of D1 dopamine receptors to regulate these behaviors. Finally, a quantitative analysis of genes examined herein revealed that C. elegans arousal thresholds were directly correlated with amount of sleep during lethargus. However, bout duration varies little and was not correlated with arousal thresholds. Conclusions: The comprehensive analysis presented here suggests that conserved genes and pathways are required for sleep in invertebrates and, likely, across the entire animal kingdom. The genetic pathway delineated in this study implicates G-alpha S and previously known genes downstream of dopamine signaling in sleep. Quantitative analysis of various components of quiescence suggests that interdependent or identical cellular and molecular mechanisms are likely to regulate both arousal and sleep entry. Citation: Singh K, Ju JY, Walsh MB, Dilorio MA, Hart AC. Deep conservation of genes required for both Drosophila melanogaster and Caenorhabditis elegans sleep includes a role for

  10. The M2 muscarinic receptors are essential for signaling in the heart left ventricle during restraint stress in mice.

    PubMed

    Tomankova, Hana; Valuskova, Paulina; Varejkova, Eva; Rotkova, Jana; Benes, Jan; Myslivecek, Jaromir

    2015-01-01

    We hypothesized that muscarinic receptors (MRs) in the heart have a role in stress responses and thus investigated changes in MR signaling (gene expression, number of receptors, adenylyl cyclase (AC), phospholipase C (PLC), protein kinase A and C (PKA and PKC) and nitric oxide synthase [NOS]) in the left ventricle, together with telemetric measurement of heart rate (HR) in mice (wild type [WT] and M2 knockout [KO]) during and after one (1R) or seven sessions (7R) of restraint stress (seven mice per group). Stress decreased M2 MR mRNA and cell surface MR in the left ventricle in WT mice. In KO mice, 1R, but not 7R, decreased surface MR. Similarly, AC activity was decreased in WT mice after 1R and 7R, whereas in KO mice, there was no change. PLC activity was also decreased after 1R in WT and KO mice. This is in accord with the concept that cAMP is a key player in HR regulation. No change was found with stress in NOS activity. Amount of AC and PKA protein was not changed, but was altered for PKC isoenzymes (PKCα, β, γ, η and ϵ (increased) in KO mice, and PKCι (increased) in WT mice). KO mice were more susceptible to stress as shown by inability to compensate HR during 120 min following repeated stress. The results imply that not only M2 but also M3 are involved in stress signaling and in allostasis. We conclude that for a normal stress response, the expression of M2 MR to mediate vagal responses is essential.

  11. Melatonin regulates somatotrope and lactotrope function through common and distinct signaling pathways in cultured primary pituitary cells from female primates.

    PubMed

    Ibáñez-Costa, Alejandro; Córdoba-Chacón, José; Gahete, Manuel D; Kineman, Rhonda D; Castaño, Justo P; Luque, Raúl M

    2015-03-01

    Melatonin (MT) is secreted by the pineal gland and exhibits a striking circadian rhythm in its release. Depending on the species studied, some pituitary hormones also display marked circadian/seasonal patterns and rhythms of secretion. However, the precise relationship between MT and pituitary function remains controversial, and studies focusing on the direct role of MT in normal pituitary cells are limited to nonprimate species. Here, adult normal primate (baboons) primary pituitary cell cultures were used to determine the direct impact of MT on the functioning of all pituitary cell types from the pars distalis. MT increased GH and prolactin (PRL) expression/release in a dose- and time-dependent fashion, a response that was blocked by somatostatin. However, MT did not significantly affect ACTH, FSH, LH, or TSH expression/release. MT did not alter GHRH- or ghrelin-induced GH and/or PRL secretions, suggesting that MT may activate similar signaling pathways as ghrelin/GHRH. The effects of MT on GH/PRL release, which are likely mediated through MT1 receptor, involve both common (adenylyl cyclase/protein kinase A/extracellular calcium-channels) and distinct (phospholipase C/intracellular calcium-channels) signaling pathways. Actions of MT on pituitary cells also included regulation of the expression of other key components for the control of somatotrope/lactotrope function (GHRH, ghrelin, and somatostatin receptors). These results show, for the first time in a primate model, that MT directly regulates somatotrope/lactotrope function, thereby lending support to the notion that the actions of MT on these cells might substantially contribute to the define daily patterns of GH and PRL observed in primates and perhaps in humans.

  12. Signaling pathways involved in human sperm hyperactivated motility stimulated by Zn2.

    PubMed

    Allouche-Fitoussi, Deborah; Bakhshi, Danit; Breitbart, Haim

    2018-05-11

    To fertilize the egg, sperm cells must reside in the female reproductive tract for several hours during which they undergo chemical and motility changes collectively called capacitation. During capacitation, the sperm develop a unique type of motility known as hyper-activated motility (HAM). The semen contains Zn 2+ in millimolar concentrations, whereas in the female reproductive tract the concentration is around 1 µM. In this study, we characterize the role of Zn 2+ in human sperm capacitation focusing on its effect on HAM. Western blot analysis revealed the presence of GPR39 type Zn-receptor localized mainly in the sperm tail. Zn 2+ at micromolar concentration stimulates HAM which is mediated by a cascade involving GPR39-Adenylyl Cyclase (AC)-cAMP-PKA-Src-EGFR and phospholipase C (PLC). Both the trans-membrane AC and the soluble-AC are involved in the stimulation of HAM by Zn 2+ . The development of HAM is precisely regulated by cAMP, in which relatively low concentration (5-10 µM) stimulated HAM, whereas at 30 µM no stimulation occurred. A similar response was seen when different concentrations of Zn 2+ were added to the cells; low Zn 2+ stimulated HAM, whereas at relatively high Zn 2+ , no effect was seen. We further demonstrate that the Ca 2+ -channel CatSper involved in Zn 2+ - stimulated HAM. These data support a role for extracellular Zn 2+ acting via GPR39 to regulate signaling pathways in sperm capacitation, leading to HAM induction. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  13. Modification of adenylate cyclase by photoaffinity analogs of forskolin

    SciTech Connect

    Ho, L.T.; Nie, Z.M.; Mende, T.J.

    1989-01-01

    Photoaffinity labeling analogs of the adenylate cyclase activator forskolin (PF) have been synthesized, purified and tested for their effect on preparations of membrane-bound, Lubrol solubilized and forskolin affinity-purified adenylate cyclase (AC). All analogs of forskolin significantly activated AC. However, in the presence of 0.1 to 0.3 microM forskolin, the less active forskolin photoaffinity probes at 100 microM caused inhibition. This inhibition was dose-dependent for PF, suggesting that PF may complete with F for the same binding site(s). After cross-linking (125I)PF-M to either membrane or Lubrol-solubilized AC preparations by photolysis, a radiolabeled 100-110 kDa protein band was observed after autoradiography followingmore » SDS-PAGE. F at 100 microM blocked the photoradiolabeling of this protein. Radioiodination of forskolin-affinity purified AC showed several protein bands on autoradiogram, however, only one band (Mr = 100-110 kDa) was specifically labeled by (125I)PF-M following photolysis. The photoaffinity-labeled protein of 100-110 kDa of AC preparation of rat adipocyte may be the catalytic unit of adenylate cyclase of rat adipocyte itself as supported by the facts that (a) no other AC-regulatory proteins are known to be of this size, (b) the catalytic unit of bovine brain enzyme is in the same range and (c) this PF specifically stimulates AC activity when assayed alone, and weekly inhibits forskolin-activation of cyclase. These studies indicate that radiolabeled PF probes may be useful for photolabeling and detecting the catalytic unit of adenylate cyclase.« less

  14. Involvement of hippocampal cAMP/cAMP-dependent protein kinase signaling pathways in a late memory consolidation phase of aversively motivated learning in rats

    PubMed Central

    Bernabeu, Ramon; Bevilaqua, Lia; Ardenghi, Patricia; Bromberg, Elke; Schmitz, Paulo; Bianchin, Marino; Izquierdo, Ivan; Medina, Jorge H.

    1997-01-01

    cAMP/cAMP-dependent protein kinase (PKA) signaling pathway has been recently proposed to participate in both the late phase of long term potentiation in the hippocampus and in the late, protein synthesis-dependent phase of memory formation. Here we report that a late memory consolidation phase of an inhibitory avoidance learning is regulated by an hippocampal cAMP signaling pathway that is activated, at least in part, by D1/D5 receptors. Bilateral infusion of SKF 38393 (7.5 μg/side), a D1/D5 receptor agonist, into the CA1 region of the dorsal hippocampus, enhanced retention of a step-down inhibitory avoidance when given 3 or 6 h, but not immediately (0 h) or 9 h, after training. In contrast, full retrograde amnesia was obtained when SCH 23390 (0.5 μg/side), a D1/D5 receptor antagonist, was infused into the hippocampus 3 or 6 h after training. Intrahippocampal infusion of 8Br-cAMP (1.25 μg/side), or forskolin (0.5 μg/side), an activator of adenylyl cyclase, enhanced memory when given 3 or 6 h after training. KT5720 (0.5 μg/side), a specific inhibitor of PKA, hindered memory consolidation when given immediately or 3 or 6 h posttraining. Rats submitted to the avoidance task showed learning-specific increases in hippocampal 3H-SCH 23390 binding and in the endogenous levels of cAMP 3 and 6 h after training. In addition, PKA activity and P-CREB (phosphorylated form of cAMP responsive element binding protein) immunoreactivity increased in the hippocampus immediately and 3 and 6 h after training. Together, these findings suggest that the late phase of memory consolidation of an inhibitory avoidance is modulated cAMP/PKA signaling pathways in the hippocampus. PMID:9192688

  15. Guanylyl Cyclase C Hormone Axis at the Intersection of Obesity and Colorectal Cancer.

    PubMed

    Blomain, Erik S; Merlino, Dante J; Pattison, Amanda M; Snook, Adam E; Waldman, Scott A

    2016-09-01

    Obesity has emerged as a principal cause of mortality worldwide, reflecting comorbidities including cancer risk, particularly in colorectum. Although this relationship is established epidemiologically, molecular mechanisms linking colorectal cancer and obesity continue to be refined. Guanylyl cyclase C (GUCY2C), a membrane-bound guanylyl cyclase expressed in intestinal epithelial cells, binds the paracrine hormones guanylin and uroguanylin, inducing cGMP signaling in colorectum and small intestine, respectively. Guanylin is the most commonly lost gene product in sporadic colorectal cancer, and its universal loss early in transformation silences GUCY2C, a tumor suppressor, disrupting epithelial homeostasis underlying tumorigenesis. In small intestine, eating induces endocrine secretion of uroguanylin, the afferent limb of a novel gut-brain axis that activates hypothalamic GUCY2C-cGMP signaling mediating satiety opposing obesity. Recent studies revealed that diet-induced obesity suppressed guanylin and uroguanylin expression in mice and humans. Hormone loss reflects reversible calorie-induced endoplasmic reticulum stress and the associated unfolded protein response, rather than the endocrine, adipokine, or inflammatory milieu of obesity. Loss of intestinal uroguanylin secretion silences the hypothalamic GUCY2C endocrine axis, creating a feed-forward loop contributing to hyperphagia in obesity. Importantly, calorie-induced guanylin loss silences the GUCY2C-cGMP paracrine axis underlying obesity-induced epithelial dysfunction and colorectal tumorigenesis. Indeed, genetically enforced guanylin replacement eliminated diet-induced intestinal tumorigenesis in mice. Taken together, these observations suggest that GUCY2C hormone axes are at the intersection of obesity and colorectal cancer. Moreover, they suggest that hormone replacement that restores GUCY2C signaling may be a novel therapeutic paradigm to prevent both hyperphagia and intestinal tumorigenesis in obesity

  16. Guanylyl Cyclase C Hormone Axis at the Intersection of Obesity and Colorectal Cancer

    PubMed Central

    Blomain, Erik S.; Merlino, Dante J.; Pattison, Amanda M.; Snook, Adam E.

    2016-01-01

    Obesity has emerged as a principal cause of mortality worldwide, reflecting comorbidities including cancer risk, particularly in colorectum. Although this relationship is established epidemiologically, molecular mechanisms linking colorectal cancer and obesity continue to be refined. Guanylyl cyclase C (GUCY2C), a membrane-bound guanylyl cyclase expressed in intestinal epithelial cells, binds the paracrine hormones guanylin and uroguanylin, inducing cGMP signaling in colorectum and small intestine, respectively. Guanylin is the most commonly lost gene product in sporadic colorectal cancer, and its universal loss early in transformation silences GUCY2C, a tumor suppressor, disrupting epithelial homeostasis underlying tumorigenesis. In small intestine, eating induces endocrine secretion of uroguanylin, the afferent limb of a novel gut-brain axis that activates hypothalamic GUCY2C-cGMP signaling mediating satiety opposing obesity. Recent studies revealed that diet-induced obesity suppressed guanylin and uroguanylin expression in mice and humans. Hormone loss reflects reversible calorie-induced endoplasmic reticulum stress and the associated unfolded protein response, rather than the endocrine, adipokine, or inflammatory milieu of obesity. Loss of intestinal uroguanylin secretion silences the hypothalamic GUCY2C endocrine axis, creating a feed-forward loop contributing to hyperphagia in obesity. Importantly, calorie-induced guanylin loss silences the GUCY2C-cGMP paracrine axis underlying obesity-induced epithelial dysfunction and colorectal tumorigenesis. Indeed, genetically enforced guanylin replacement eliminated diet-induced intestinal tumorigenesis in mice. Taken together, these observations suggest that GUCY2C hormone axes are at the intersection of obesity and colorectal cancer. Moreover, they suggest that hormone replacement that restores GUCY2C signaling may be a novel therapeutic paradigm to prevent both hyperphagia and intestinal tumorigenesis in obesity

  17. The cAMP signaling system inhibits the repair of γ-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells.

    PubMed

    Cho, Eun-Ah; Juhnn, Yong-Sung

    2012-06-01

    Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on γ-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (GαsQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of GαsQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after γ-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2'-O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2'-O-Me-cAMP and restored XRCC1 protein level following γ-ray irradiation. From these results, we conclude that the cAMP signaling system inhibits the repair of γ-ray-induced DNA damage by promoting the ubiquitin-proteasome dependent degradation of XRCC1 in an Epac-dependent pathway in lung cancer cells. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Fetal nicotine exposure produces postnatal up-regulation of adenylate cyclase activity in peripheral tissues

    SciTech Connect

    Slotkin, T.A.; Navarro, H.A.; McCook, E.C.

    1990-01-01

    Gestational exposure to nicotine has been shown to affect development of noradrenergic activity in both the central and peripheral nervous systems. In the current study, pregnant rats received nicotine infusions of 6 mg/kg/day throughout gestation, administered by osmotic minipump implants. After birth, offspring of the nicotine-infused dams exhibited marked increases in basal adenylate cyclase activity in membranes prepared from kidney and heart, as well as supersensitivity to stimulation by either a {beta}-adrenergic agonist, isoproterenol, or by forskolin. The altered responses were not accompanied by up-regulation of {beta}-adrenergic receptors: in fact, ({sup 125}I)pindolol binding was significantly decreased in the nicotine group.more » These results indicate that fetal nicotine exposure affects enzymes involved in membrane receptor signal transduction, leading to altered responsiveness independently of changes at the receptor level.« less

  19. GCPred: a web tool for guanylyl cyclase functional centre prediction from amino acid sequence.

    PubMed

    Xu, Nuo; Fu, Dongfang; Li, Shiang; Wang, Yuxuan; Wong, Aloysius

    2018-06-15

    GCPred is a webserver for the prediction of guanylyl cyclase (GC) functional centres from amino acid sequence. GCs are enzymes that generate the signalling molecule cyclic guanosine 3', 5'-monophosphate from guanosine-5'-triphosphate. A novel class of GC centres (GCCs) has been identified in complex plant proteins. Using currently available experimental data, GCPred is created to automate and facilitate the identification of similar GCCs. The server features GCC values that consider in its calculation, the physicochemical properties of amino acids constituting the GCC and the conserved amino acids within the centre. From user input amino acid sequence, the server returns a table of GCC values and graphs depicting deviations from mean values. The utility of this server is demonstrated using plant proteins and the human interleukin-1 receptor-associated kinase family of proteins as example. The GCPred server is available at http://gcpred.com. Supplementary data are available at Bioinformatics online.

  20. High skeletal muscle adenylate cyclase in malignant hyperthermia.

    PubMed Central

    Willner, J H; Cerri, C G; Wood, D S

    1981-01-01

    Malignant hyperthermia occurs in humans with several congenital myopathies, usually in response to general anesthesia. Commonly, individuals who develop this syndrome lack symptoms of muscle disease, and their muscle lacks specific pathological changes. A biochemical marker for this myopathy has not previously been available; we found activity of adenylate cyclase and content of cyclic AMP to be abnormally high in skeletal muscle. Secondary modification of protein phosphorylation could explain observed abnormalities of phosphorylase activation and sarcoplasmic reticulum function. PMID:6271806

  1. Porcine CD38 exhibits prominent secondary NAD(+) cyclase activity.

    PubMed

    Ting, Kai Yiu; Leung, Christina F P; Graeff, Richard M; Lee, Hon Cheung; Hao, Quan; Kotaka, Masayo

    2016-03-01

    Cyclic ADP-ribose (cADPR) mobilizes intracellular Ca(2+) stores and activates Ca(2+) influx to regulate a wide range of physiological processes. It is one of the products produced from the catalysis of NAD(+) by the multifunctional CD38/ADP-ribosyl cyclase superfamily. After elimination of the nicotinamide ring by the enzyme, the reaction intermediate of NAD(+) can either be hydrolyzed to form linear ADPR or cyclized to form cADPR. We have previously shown that human CD38 exhibits a higher preference towards the hydrolysis of NAD(+) to form linear ADPR while Aplysia ADP-ribosyl cyclase prefers cyclizing NAD(+) to form cADPR. In this study, we characterized the enzymatic properties of porcine CD38 and revealed that it has a prominent secondary NAD(+) cyclase activity producing cADPR. We also determined the X-ray crystallographic structures of porcine CD38 and were able to observe conformational flexibility at the base of the active site of the enzyme which allow the NAD(+) reaction intermediate to adopt conformations resulting in both hydrolysis and cyclization forming linear ADPR and cADPR respectively. © 2016 The Protein Society.

  2. Noncanonical Gβ Gib2 is a scaffolding protein promoting cAMP signaling through functions of Ras1 and Cac1 proteins in Cryptococcus neoformans.

    PubMed

    Wang, Yanli; Shen, Gui; Gong, Jinjun; Shen, Danyu; Whittington, Amy; Qing, Jiang; Treloar, Joshua; Boisvert, Scott; Zhang, Zhengguang; Yang, Cai; Wang, Ping

    2014-05-02

    Gβ-like/RACK1 functions as a key mediator of various pathways and contributes to numerous cellular functions in eukaryotic organisms. In the pathogenic fungus Cryptococcus neoformans, noncanonical Gβ Gib2 promotes cAMP signaling in cells lacking normal Gpa1 function while displaying versatility in interactions with Gα Gpa1, protein kinase Pkc1, and endocytic intersectin Cin1. To elucidate the Gib2 functional mechanism(s), we demonstrate that Gib2 is required for normal growth and virulence. We show that Gib2 directly binds to Gpa1 and Gγ Gpg1/Gpg2 and that it interacts with phosphodiesterase Pde2 and monomeric GTPase Ras1. Pde2 remains functionally dispensable, but Ras1 is found to associate with adenylyl cyclase Cac1 through the conserved Ras association domain. In addition, the ras1 mutant exhibits normal capsule formation, whereas the ras1 gpa1 mutant displays enhanced capsule formation, and the ras1 gpa1 cac1 mutant is acapsular. Collectively, these findings suggest that Gib2 promotes cAMP levels by relieving an inhibitory function of Ras1 on Cac1 in the absence of Gpa1. In addition, using GST affinity purification combined with mass spectrometry, we identified 47 additional proteins that interact with Gib2. These proteins have putative functions ranging from signal transduction, energy generation, metabolism, and stress response to ribosomal function. After establishing and validating a protein-protein interactive network, we believe Gib2 to be a key adaptor/scaffolding protein that drives the formation of various protein complexes required for growth and virulence. Our study reveals Gib2 as an essential component in deciphering the complexity of regulatory networks that control growth and virulence in C. neoformans.

  3. Forskolin increases angiogenesis through the coordinated cross-talk of PKA-dependent VEGF expression and Epac-mediated PI3K/Akt/eNOS signaling.

    PubMed

    Namkoong, Seung; Kim, Chun-Ki; Cho, Young-Lai; Kim, Ji-Hee; Lee, Hansoo; Ha, Kwon-Soo; Choe, Jongseon; Kim, Pyeung-Hyeun; Won, Moo-Ho; Kwon, Young-Geun; Shim, Eun Bo; Kim, Young-Myeong

    2009-06-01

    Forskolin, a potent activator of adenylyl cyclases, has been implicated in modulating angiogenesis, but the underlying mechanism has not been clearly elucidated. We investigated the signal mechanism by which forskolin regulates angiogenesis. Forskolin stimulated angiogenesis of human endothelial cells and in vivo neovascularization, which was accompanied by phosphorylation of CREB, ERK, Akt, and endothelial nitric oxide synthase (eNOS) as well as NO production and VEGF expression. Forskolin-induced CREB phosphorylation, VEGF promoter activity, and VEGF expression were blocked by the PKA inhibitor PKI.Moreover, phosphorylation of ERK by forskolin was inhibited by the MEK inhibitor PD98059, but not PKI. The forskolin-induced Akt/eNOS/NO pathway was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, but not significantly suppressed by PKI. These inhibitors and a NOS inhibitor partially inhibited forskolin-induced angiogenesis. The exchange protein directly activated by cAMP (Epac) activator, 8CPT-2Me-cAMP, promoted the Akt/eNOS/NO pathway and ERK phosphorylation,but did not induce CREB phosphorylation and VEGF expression. The angiogenic effect of the Epac activator was diminished by the inhibition of PI3K and MEK, but not by the PKA inhibitor. Small interfering RNA-mediated knockdown of Epac1 suppressed forskolin-induced angiogenesis and phosphorylation of ERK, Akt, and eNOS, but not CREB phosphorylation and VEGF expression. These results suggest that forskolin stimulates angiogenesis through coordinated cross-talk between two distinct pathways, PKA-dependent VEGF expression and Epac-dependent ERKactivation and PI3K/Akt/eNOS/NO signaling.

  4. A possible mechanism for improvement by a cognition-enhancer nefiracetam of spatial memory function and cAMP-mediated signal transduction system in sustained cerebral ischaemia in rats

    PubMed Central

    Takeo, Satoshi; Niimura, Makiko; Miyake-Takagi, Keiko; Nagakura, Akira; Fukatsu, Tomoko; Ando, Tsuyoshi; Takagi, Norio; Tanonaka, Kouichi; Hara, Junko

    2003-01-01

    Accumulated evidence indicates that the adenylyl cyclase (AC)/cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)/cAMP-responsive element binding protein (CREB) signal transduction system may be linked to learning and memory function. The effects of nefiracetam, which has been developed as a cognition enhancer, on spatial memory function and the AC/cAMP/PKA/CREB signal transduction system in rats with sustained cerebral ischaemia were examined. Microsphere embolism (ME)-induced sustained cerebral ischaemia was produced by injection of 700 microspheres (48 μm in diameter) into the right hemisphere of rats. Daily oral administration of nefiracetam (10 mg kg−1 day−1) was started from 15 h after the operation. The delayed treatment with nefiracetam attenuated the ME-induced prolongation of the escape latency in the water maze task that was examined on day 7 to 9 after ME, but it did not reduce the infarct size. ME decreased Ca2+/calmodulin (CaM)-stimulated AC (AC-I) activity, cAMP content, cytosolic PKA Cβ level, nuclear PKA Cα and Cβ levels, and reduced the phosphorylation and DNA-binding activity of CREB in the nucleus in the right parietal cortex and hippocampus on day 3 after ME. The ME-induced changes in these variables did not occur by the delayed treatment with nefiracetam. These results suggest that nefiracetam preserved cognitive function, or prevented cognitive dysfunction, after sustained cerebral ischaemia and that the effect is, in part, attributable to the prevention of the ischaemia-induced impairment of the AC/cAMP/PKA/CREB signal transduction pathway. PMID:12598418

  5. Identification of terminal adenylyl transferase activity of the poliovirus polymerase 3Dpol.

    PubMed Central

    Neufeld, K L; Galarza, J M; Richards, O C; Summers, D F; Ehrenfeld, E

    1994-01-01

    A terminal adenylyl transferase (TATase) activity has been identified in preparations of purified poliovirus RNA-dependent RNA polymerase (3Dpol). Highly purified 3Dpol is capable of adding [32P]AMP to the 3' ends of chemically synthesized 12-nucleotide (nt)-long RNAs. The purified 52-kDa polypeptide, isolated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renatured, retained the TATase activity. Two 3Dpol mutants, purified from Escherichia coli expression systems, displayed no detectable polymerase activity and were unable to catalyze TATase activity. Likewise, extracts from the parental E. coli strain that harbored no expression plasmid were unable to catalyze formation of the TATase products. With the RNA oligonucleotide 5'-CCUGCUUUUGCA-3' used as an acceptor, the products formed by wild-type 3Dpol were 9 and 18 nt longer than the 12-nt oligomer. GTP, CTP, and UTP did not serve as substrates for transfer to this RNA, either by themselves or when all deoxynucleoside triphosphates were present in the reaction. Results from kinetic and stoichiometric analyses suggest that the reaction is catalytic and shows substrate and enzyme dependence. The 3'-terminal 13 nt of poliovirus minus-strand RNA also served as an acceptor for TATase activity, raising the possibility that this activity functions in poliovirus RNA replication. The efficiency of utilization and the nature of the products formed during the reaction were dependent on the acceptor RNA. Images PMID:8057462

  6. Crystal structure of papaya glutaminyl cyclase, an archetype for plant and bacterial glutaminyl cyclases.

    PubMed

    Wintjens, René; Belrhali, Hassan; Clantin, Bernard; Azarkan, Mohamed; Bompard, Coralie; Baeyens-Volant, Danielle; Looze, Yvan; Villeret, Vincent

    2006-03-24

    Glutaminyl cyclases (QCs) (EC 2.3.2.5) catalyze the intramolecular cyclization of protein N-terminal glutamine residues into pyroglutamic acid with the concomitant liberation of ammonia. QCs may be classified in two groups containing, respectively, the mammalian enzymes, and the enzymes from plants, bacteria, and parasites. The crystal structure of the QC from the latex of Carica papaya (PQC) has been determined at 1.7A resolution. The structure was solved by the single wavelength anomalous diffraction technique using sulfur and zinc as anomalous scatterers. The enzyme folds into a five-bladed beta-propeller, with two additional alpha-helices and one beta hairpin. The propeller closure is achieved via an original molecular velcro, which links the last two blades into a large eight stranded beta-sheet. The zinc ion present in the PQC is bound via an octahedral coordination into an elongated cavity located along the pseudo 5-fold axis of the beta-propeller fold. This zinc ion presumably plays a structural role and may contribute to the exceptional stability of PQC, along with an extended hydrophobic packing, the absence of long loops, the three-joint molecular velcro and the overall folding itself. Multiple sequence alignments combined with structural analyses have allowed us to tentatively locate the active site, which is filled in the crystal structure either by a Tris molecule or an acetate ion. These analyses are further supported by the experimental evidence that Tris is a competitive inhibitor of PQC. The active site is located at the C-terminal entrance of the PQC central tunnel. W83, W110, W169, Q24, E69, N155, K225, F22 and F67 are highly conserved residues in the C-terminal entrance, and their putative role in catalysis is discussed. The PQC structure is representative of the plants, bacterial and parasite enzymes and contrasts with that of mammalian enzymes, that may possibly share a conserved scaffold of the bacterial aminopeptidase.

  7. Guanylate cyclase-activating protein 2 contributes to phototransduction and light adaptation in mouse cone photoreceptors.

    PubMed

    Vinberg, Frans; Peshenko, Igor V; Chen, Jeannie; Dizhoor, Alexander M; Kefalov, Vladimir J

    2018-05-11

    Light adaptation of photoreceptor cells is mediated by Ca 2+ -dependent mechanisms. In darkness, Ca 2+ influx through cGMP-gated channels into the outer segment of photoreceptors is balanced by Ca 2+ extrusion via Na + /Ca 2+ , K + exchangers (NCKXs). Light activates a G protein signaling cascade, which closes cGMP-gated channels and decreases Ca 2+ levels in photoreceptor outer segment because of continuing Ca 2+ extrusion by NCKXs. Guanylate cyclase-activating proteins (GCAPs) then up-regulate cGMP synthesis by activating retinal membrane guanylate cyclases (RetGCs) in low Ca 2+ This activation of RetGC accelerates photoresponse recovery and critically contributes to light adaptation of the nighttime rod and daytime cone photoreceptors. In mouse rod photoreceptors, GCAP1 and GCAP2 both contribute to the Ca 2+ -feedback mechanism. In contrast, only GCAP1 appears to modulate RetGC activity in mouse cones because evidence of GCAP2 expression in cones is lacking. Surprisingly, we found that GCAP2 is expressed in cones and can regulate light sensitivity and response kinetics as well as light adaptation of GCAP1-deficient mouse cones. Furthermore, we show that GCAP2 promotes cGMP synthesis and cGMP-gated channel opening in mouse cones exposed to low Ca 2+ Our biochemical model and experiments indicate that GCAP2 significantly contributes to the activation of RetGC1 at low Ca 2+ when GCAP1 is not present. Of note, in WT mouse cones, GCAP1 dominates the regulation of cGMP synthesis. We conclude that, under normal physiological conditions, GCAP1 dominates the regulation of cGMP synthesis in mouse cones, but if its function becomes compromised, GCAP2 contributes to the regulation of phototransduction and light adaptation of cones. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Oxygen Binding and Redox Properties of the Heme in Soluble Guanylate Cyclase

    PubMed Central

    Makino, Ryu; Park, Sam-yon; Obayashi, Eiji; Iizuka, Tetsutaro; Hori, Hiroshi; Shiro, Yoshitugu

    2011-01-01

    Soluble guanylate cyclase is an NO-sensing hemoprotein that serves as a NO receptor in NO-mediated signaling pathways. It has been believed that this enzyme displays no measurable affinity for O2, thereby enabling the selective NO sensing in aerobic environments. Despite the physiological significance, the reactivity of the enzyme-heme for O2 has not been examined in detail. In this paper we demonstrated that the high spin heme of the ferrous enzyme converted to a low spin oxyheme (Fe2+-O2) when frozen at 77 K in the presence of O2. The ligation of O2 was confirmed by EPR analyses using cobalt-substituted enzyme. The oxy form was produced also under solution conditions at −7 °C, with the extremely low affinity for O2. The low O2 affinity was not caused by a distal steric protein effect and by rupture of the Fe2+-proximal His bond as revealed by extended x-ray absorption fine structure. The midpoint potential of the enzyme-heme was +187 mV, which is the most positive among high spin protoheme-hemoproteins. This observation implies that the electron density of the ferrous heme iron is relatively low by comparison to those of other hemoproteins, presumably due to the weak Fe2+-proximal His bond. Based on our results, we propose that the weak Fe2+-proximal His bond is a key determinant for the low O2 affinity of the heme moiety of soluble guanylate cyclase. PMID:21385878

  9. Deinococcus radiodurans RNA ligase exemplifies a novel ligase clade with a distinctive N-terminal module that is important for 5'-PO4 nick sealing and ligase adenylylation but dispensable for phosphodiester formation at an adenylylated nick.

    PubMed

    Raymond, Amy; Shuman, Stewart

    2007-01-01

    Deinococcus radiodurans RNA ligase (DraRnl) is a template-directed ligase that seals nicked duplexes in which the 3'-OH strand is RNA. DraRnl is a 342 amino acid polypeptide composed of a C-terminal adenylyltransferase domain fused to a distinctive 126 amino acid N-terminal module (a putative OB-fold). An alanine scan of the C domain identified 9 amino acids essential for nick ligation, which are located within nucleotidyltransferase motifs I, Ia, III, IIIa, IV and V. Seven mutants were dysfunctional by virtue of defects in ligase adenylylation: T163A, H167A, G168A, K186A, E230A, F281A and E305A. Four of these were also defective in phosphodiester formation at a preadenylylated nick: G168A, E230A, F281A and E305A. Two nick sealing-defective mutants were active in ligase adenylylation and sealing a preadenylylated nick, thereby implicating Ser185 and Lys326 in transfer of AMP from the enzyme to the nick 5'-PO(4). Whereas deletion of the N-terminal domain suppressed overall nick ligation and ligase adenylylation, it did not compromise sealing at a preadenylylated nick. Mutational analysis of 15 residues of the N domain identified Lys26, Gln31 and Arg79 as key constituents. Structure-activity relationships at the essential residues were determined via conservative substitutions. We propose that DraRnl typifies a new clade of polynucleotide ligases. DraRnl homologs are detected in several eukaryal proteomes.

  10. Deinococcus radiodurans RNA ligase exemplifies a novel ligase clade with a distinctive N-terminal module that is important for 5′-PO4 nick sealing and ligase adenylylation but dispensable for phosphodiester formation at an adenylylated nick

    PubMed Central

    Raymond, Amy; Shuman, Stewart

    2007-01-01

    Deinococcus radiodurans RNA ligase (DraRnl) is a template-directed ligase that seals nicked duplexes in which the 3′-OH strand is RNA. DraRnl is a 342 amino acid polypeptide composed of a C-terminal adenylyltransferase domain fused to a distinctive 126 amino acid N-terminal module (a putative OB-fold). An alanine scan of the C domain identified 9 amino acids essential for nick ligation, which are located within nucleotidyltransferase motifs I, Ia, III, IIIa, IV and V. Seven mutants were dysfunctional by virtue of defects in ligase adenylylation: T163A, H167A, G168A, K186A, E230A, F281A and E305A. Four of these were also defective in phosphodiester formation at a preadenylylated nick: G168A, E230A, F281A and E305A. Two nick sealing-defective mutants were active in ligase adenylylation and sealing a preadenylylated nick, thereby implicating Ser185 and Lys326 in transfer of AMP from the enzyme to the nick 5′-PO4. Whereas deletion of the N-terminal domain suppressed overall nick ligation and ligase adenylylation, it did not compromise sealing at a preadenylylated nick. Mutational analysis of 15 residues of the N domain identified Lys26, Gln31 and Arg79 as key constituents. Structure–activity relationships at the essential residues were determined via conservative substitutions. We propose that DraRnl typifies a new clade of polynucleotide ligases. DraRnl homologs are detected in several eukaryal proteomes. PMID:17204483

  11. A new small molecule inhibitor of soluble guanylate cyclase

    PubMed Central

    Mota, Filipa; Gane, Paul; Hampden-Smith, Kathryn; Allerston, Charles K.; Garthwaite, John; Selwood, David L.

    2015-01-01

    Soluble guanylate cyclase (sGC) is a haem containing enzyme that regulates cardiovascular homeostasis and multiple mechanisms in the central and peripheral nervous system. Commonly used inhibitors of sGC activity act through oxidation of the haem moiety, however they also bind haemoglobin and this limits their bioavailability for in vivo studies. We have discovered a new class of small molecule inhibitors of sGC and have characterised a compound designated D12 (compound 10) which binds to the catalytic domain of the enzyme with a KD of 11 μM in a SPR assay. PMID:26264842

  12. Intestinal Enteroids Model Guanylate Cyclase C-Dependent Secretion Induced by Heat-Stable Enterotoxins.

    PubMed

    Pattison, Amanda M; Blomain, Erik S; Merlino, Dante J; Wang, Fang; Crissey, Mary Ann S; Kraft, Crystal L; Rappaport, Jeff A; Snook, Adam E; Lynch, John P; Waldman, Scott A

    2016-10-01

    Enterotoxigenic Escherichia coli (ETEC) causes ∼20% of the acute infectious diarrhea (AID) episodes worldwide, often by producing heat-stable enterotoxins (STs), which are peptides structurally homologous to paracrine hormones of the intestinal guanylate cyclase C (GUCY2C) receptor. While molecular mechanisms mediating ST-induced intestinal secretion have been defined, advancements in therapeutics have been hampered for decades by the paucity of disease models that integrate molecular and functional endpoints amenable to high-throughput screening. Here, we reveal that mouse and human intestinal enteroids in three-dimensional ex vivo cultures express the components of the GUCY2C secretory signaling axis. ST and its structural analog, linaclotide, an FDA-approved oral secretagog, induced fluid accumulation quantified simultaneously in scores of enteroid lumens, recapitulating ETEC-induced intestinal secretion. Enteroid secretion depended on canonical molecular signaling events responsible for ETEC-induced diarrhea, including cyclic GMP (cGMP) produced by GUCY2C, activation of cGMP-dependent protein kinase (PKG), and opening of the cystic fibrosis transmembrane conductance regulator (CFTR). Importantly, pharmacological inhibition of CFTR abrogated enteroid fluid secretion, providing proof of concept for the utility of this model to screen antidiarrheal agents. Intestinal enteroids offer a unique model, integrating the GUCY2C signaling axis and luminal fluid secretion, to explore the pathophysiology of, and develop platforms for, high-throughput drug screening to identify novel compounds to prevent and treat ETEC diarrheal disease. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  13. Upregulation of CREM/ICER suppresses wound endothelial CRE-HIF-1α-VEGF-dependent signaling and impairs angiogenesis in type 2 diabetes

    PubMed Central

    Bitar, Milad S.; Al-Mulla, Fahd

    2015-01-01

    Impaired angiogenesis and endothelial dysfunction in type 2 diabetes constitute dominant risk factors for non-healing wounds and most forms of cardiovascular disease. We propose that diabetes shifts the ‘angiogenic balance’ in favor of an excessive anti-angiogenic phenotype. Herein, we report that diabetes impairs in vivo sponge angiogenic capacity by decreasing VEGF expression and fibrovascular invasion, and reciprocally enhances the formation of angiostatic molecules, such as thrombospondins, NFκB and FasL. Defective in vivo angiogenesis prompted cellular studies in cultured endothelial cells derived from subcutaneous sponge implants (SIECs) of control and Goto-Kakizaki rats. Ensuing data from diabetic SIECs demonstrated a marked upregulation in cAMP-PKA-CREB signaling, possibly stemming from increased expression of adenylyl cyclase isoforms 3 and 8, and decreased expression of PDE3. Mechanistically, we found that oxidative stress and PKA activation in diabetes enhanced CREM/ICER expression. This reduces IRS2 cellular content by inhibiting cAMP response element (CRE) transcriptional activity. Consequently, a decrease in the activity of Akt-mTOR ensued with a concomitant reduction in the total and nuclear protein levels of HIF-1α. Limiting HIF-1α availability for the specific hypoxia response elements in diabetic SIECs elicited a marked reduction in VEGF expression, both at the mRNA and protein levels. These molecular abnormalities were illustrated functionally by a defect in various pro-angiogenic properties, including cell proliferation, migration and tube formation. A genetic-based strategy in diabetic SIECs using siRNAs against CREM/ICER significantly augmented the PKA-dependent VEGF expression. To this end, the current data identify the importance of CREM/ICER as a negative regulator of endothelial function and establish a link between CREM/ICER overexpression and impaired angiogenesis during the course of diabetes. Moreover, it could also point to

  14. Protein kinase-dependent oxidative regulation of the cardiac Na+–K+ pump: evidence from in vivo and in vitro modulation of cell signalling

    PubMed Central

    Galougahi, Keyvan Karimi; Liu, Chia-Chi; Garcia, Alvaro; Fry, Natasha A S; Hamilton, Elisha J; Rasmussen, Helge H; Figtree, Gemma A

    2013-01-01

    The widely reported stimulation of the cardiac Na+–K+ pump by protein kinase A (PKA) should oppose other effects of PKA to increase contractility of the normal heart. It should also reduce harmful raised myocyte Na+ levels in heart failure, yet blockade of the β1 adrenergic receptor (AR), coupled to PKA signalling, is beneficial. We treated rabbits with the β1 AR antagonist metoprolol to modulate PKA activity and studied cardiac myocytes ex vivo. Metoprolol increased electrogenic pump current (Ip) in voltage clamped myocytes and reduced glutathionylation of the β1 pump subunit, an oxidative modification causally related to pump inhibition. Activation of adenylyl cyclase with forskolin to enhance cAMP synthesis or inclusion of the catalytic subunit of PKA in patch pipette solutions abolished the increase in Ip in voltage clamped myocytes induced by treatment with metoprolol, supporting cAMP/PKA-mediated pump inhibition. Metoprolol reduced myocardial PKA and protein kinase C (PKC) activities, reduced coimmunoprecipitation of cytosolic p47phox and membranous p22phox NADPH oxidase subunits and reduced myocardial O2•−-sensitive dihydroethidium fluorescence. Treatment also enhanced coimmunoprecipitation of the β1 pump subunit with glutaredoxin 1 that catalyses de-glutathionylation. Since angiotensin II induces PKC-dependent activation of NADPH oxidase, we examined the effects of angiotensin-converting enzyme inhibition with captopril. This treatment had no effect on PKA activity but reduced the activity of PKC, reduced β1 subunit glutathionylation and increased Ip. The PKA-induced Na+–K+ pump inhibition we report should act with other mechanisms that enhance contractility of the normal heart but accentuate the harmful effects of raised cytosolic Na+ in the failing heart. This scheme is consistent with the efficacy of β1 AR blockade in the treatment of heart failure. PMID:23587884

  15. G protein-coupled estrogen receptor inhibits the P2Y receptor-mediated Ca(2+) signaling pathway in human airway epithelia.

    PubMed

    Hao, Yuan; Chow, Alison W; Yip, Wallace C; Li, Chi H; Wan, Tai F; Tong, Benjamin C; Cheung, King H; Chan, Wood Y; Chen, Yangchao; Cheng, Christopher H; Ko, Wing H

    2016-08-01

    P2Y receptor activation causes the release of inflammatory cytokines in the bronchial epithelium, whereas G protein-coupled estrogen receptor (GPER), a novel estrogen (E2) receptor, may play an anti-inflammatory role in this process. We investigated the cellular mechanisms underlying the inhibitory effect of GPER activation on the P2Y receptor-mediated Ca(2+) signaling pathway and cytokine production in airway epithelia. Expression of GPER in primary human bronchial epithelial (HBE) or 16HBE14o- cells was confirmed on both the mRNA and protein levels. Stimulation of HBE or 16HBE14o- cells with E2 or G1, a specific agonist of GPER, attenuated the nucleotide-evoked increases in [Ca(2+)]i, whereas this effect was reversed by G15, a GPER-specific antagonist. G1 inhibited the secretion of two proinflammatory cytokines, interleukin (IL)-6 and IL-8, in cells stimulated by adenosine 5'-(γ-thio)triphosphate (ATPγS). G1 stimulated a real-time increase in cAMP levels in 16HBE14o- cells, which could be inhibited by adenylyl cyclase inhibitors. The inhibitory effects of E2 or G1 on P2Y receptor-induced increases in Ca(2+) were reversed by treating the cells with a protein kinase A (PKA) inhibitor. These results demonstrated that the inhibitory effects of G1 or E2 on P2Y receptor-mediated Ca(2+) mobilization and cytokine secretion were due to GPER-mediated activation of a cAMP-dependent PKA pathway. This study has reported, for the first time, the expression and function of GPER as an anti-inflammatory component in human bronchial epithelia, which may mediate through its opposing effects on the pro-inflammatory pathway activated by the P2Y receptors in inflamed airway epithelia.

  16. Activation of Extracellular Signal-Regulated Kinase in the Anterior Cingulate Cortex Contributes to the Induction and Expression of Affective Pain

    PubMed Central

    Cao, Hong; Gao, Yong-Jing; Ren, Wen-Hua; Li, Ting-Ting; Duan, Kai-Zheng; Cui, Yi-Hui; Cao, Xiao-Hua; Zhao, Zhi-Qi; Ji, Ru-Rong; Zhang, Yu-Qiu

    2009-01-01

    The anterior cingulate cortex (ACC) is implicated in the affective response to noxious stimuli. However, little is known about the molecular mechanisms involved. The present study demonstrated that extracellular signal-regulated kinase (ERK) activation in the ACC plays a crucial role in pain-related negative emotion. Intraplantar formalin injection produced a transient ERK activation in laminae V–VI and a persistent ERK activation in laminae II–III of the rostral ACC (rACC) bilaterally. Using formalin-induced conditioned place avoidance (F-CPA) in rats, which is believed to reflect the pain-related negative emotion, we found that blockade of ERK activation in the rACC with MEK inhibitors prevented the induction of F-CPA. Interestingly, this blockade did not affect formalin-induced two-phase spontaneous nociceptive responses and CPA acquisition induced by electric foot-shock or U69,593, an innocuous aversive agent. Upstream, NMDA receptor, adenylyl cyclase (AC) and PKA activators activated ERK in rACC slices. Consistently, intra-rACC microinjection of AC or PKA inhibitors prevented F-CPA induction. Downstream, phosphorylation of cAMP response element binding protein (CREB) was induced in the rACC by formalin injection and by NMDA, AC and PKA activators in brain slices, which was suppressed by MEK inhibitors. Furthermore, ERK also contributed to the expression of pain-related negative emotion. Thus, when rats were re-exposed to the conditioning context for retrieval of pain experience, ERK and CREB were re-activated in the rACC, and inhibiting ERK activation blocked the expression of F-CPA. All together, our results demonstrate that ERK activation in the rACC is required for the induction and expression of pain-related negative affect. PMID:19279268

  17. Relationship of calcium and membrane guanylate cyclase in adrenocorticotropin-induced steroidogenesis.

    PubMed

    Nambi, P; Aiyar, N V; Roberts, A N; Sharma, R K

    1982-07-01

    Chlorpromazine, when incubated with isolated adrenal cells, inhibited the ACTH-stimulated formation of cGMP and corticosterone production. It also inhibited the ACTH-stimulated membrane guanylate cyclase, but did not affect the binding of ACTH to the membrane receptors. cGMP-induced steroidogenesis was not affected by the drug. These data indicate that chlorpromazine interferes with adrenal steroid metabolism at a site between the hormone receptor and guanylate cyclase and also show that guanylate cyclase is composed of separate receptor and catalytic components. Furthermore, based on the premise that chlorpromazine exerts its inhibitory action by blocking the binding of a calcium receptor protein, such as calmodulin, to the receptor-coupled guanylate cyclase, it is proposed that the interaction of calcium, presumably through a calcium-binding protein, is essential for ACTH-dependent guanylate cyclase.

  18. Structural analysis of an oxygen-regulated diguanylate cyclase.

    PubMed

    Tarnawski, Miroslaw; Barends, Thomas R M; Schlichting, Ilme

    2015-11-01

    Cyclic di-GMP is a bacterial second messenger that is involved in switching between motile and sessile lifestyles. Given the medical importance of biofilm formation, there has been increasing interest in understanding the synthesis and degradation of cyclic di-GMPs and their regulation in various bacterial pathogens. Environmental cues are detected by sensing domains coupled to GGDEF and EAL or HD-GYP domains that have diguanylate cyclase and phosphodiesterase activities, respectively, producing and degrading cyclic di-GMP. The Escherichia coli protein DosC (also known as YddV) consists of an oxygen-sensing domain belonging to the class of globin sensors that is coupled to a C-terminal GGDEF domain via a previously uncharacterized middle domain. DosC is one of the most strongly expressed GGDEF proteins in E. coli, but to date structural information on this and related proteins is scarce. Here, the high-resolution structural characterization of the oxygen-sensing globin domain, the middle domain and the catalytic GGDEF domain in apo and substrate-bound forms is described. The structural changes between the iron(III) and iron(II) forms of the sensor globin domain suggest a mechanism for oxygen-dependent regulation. The structural information on the individual domains is combined into a model of the dimeric DosC holoprotein. These findings have direct implications for the oxygen-dependent regulation of the activity of the cyclase domain.

  19. Membrane guanylate cyclase, a multimodal transduction machine: history, present, and future directions

    PubMed Central

    Sharma, Rameshwar K.; Duda, Teresa

    2014-01-01

    A sequel to these authors' earlier comprehensive reviews which covered the field of mammalian membrane guanylate cyclase (MGC) from its origin to the year 2010, this article contains 13 sections. The first is historical and covers MGC from the year 1963–1987, summarizing its colorful developmental stages from its passionate pursuit to its consolidation. The second deals with the establishment of its biochemical identity. MGC becomes the transducer of a hormonal signal and founder of the peptide hormone receptor family, and creates the notion that hormone signal transduction is its sole physiological function. The third defines its expansion. The discovery of ROS-GC subfamily is made and it links ROS-GC with the physiology of phototransduction. Sections ROS-GC, a Ca2+-Modulated Two Component Transduction System to Migration Patterns and Translations of the GCAP Signals Into Production of Cyclic GMP are Different cover its biochemistry and physiology. The noteworthy events are that augmented by GCAPs, ROS-GC proves to be a transducer of the free Ca2+ signals generated within neurons; ROS-GC becomes a two-component transduction system and establishes itself as a source of cyclic GMP, the second messenger of phototransduction. Section ROS-GC1 Gene Linked Retinal Dystrophies demonstrates how this knowledge begins to be translated into the diagnosis and providing the molecular definition of retinal dystrophies. Section Controlled By Low and High Levels of [Ca2+]i, ROS-GC1 is a Bimodal Transduction Switch discusses a striking property of ROS-GC where it becomes a “[Ca2+]i bimodal switch” and transcends its signaling role in other neural processes. In this course, discovery of the first CD-GCAP (Ca2+-dependent guanylate cyclase activator), the S100B protein, is made. It extends the role of the ROS-GC transduction system beyond the phototransduction to the signaling processes in the synapse region between photoreceptor and cone ON-bipolar cells; in section Ca2

  20. The Importance of Sulfate Adenylyl Transferase in S and O Fractionation by Sulfate Reducing Bacteria

    NASA Astrophysics Data System (ADS)

    Smith, D. A.; Johnston, D. T.; Bradley, A. S.

    2016-12-01

    Microbial sulfate reduction (MSR) is critical to the oxidation of organic matter in modern and ancient oceans, and plays an important role in regulating the redox state of the Earth's surface. The sulfur and oxygen isotopic composition of seawater sulfate and of sulfate minerals reflect the biogeochemical processes that cycle sulfur, of which MSR is among the most important. MSR is a multi-enzymatic reaction network that partitions the isotopes of sulfur and oxygen as a consequence of both the flux of sulfate through this biochemical network and the fractionation imposed by each individual enzyme. MSR affects the δ18O of residual, extracellular sulfate mainly by the equilibration of the MSR intermediate sulfite with extracellular water (Antler et al., 2013 GCA, Wankel et al., 2013 Geobiol). A series of oxidative and exchange reactions catalyzed by APS reductase (APSr), sulfate adenylyl transferase (Sat), and sulfate transporters promote the conversion of water-equilibrated intracellular sulfite to extracellular sulfate. The flux of sulfoxy anions via these proteins will be, at least in part, dependent on the activity of these enzymes. To test this, we examined sulfur and oxygen isotope fractionation in genetically engineered mutants of the sulfate reducing bacterium Desulfovibrio vulgaris Hildenborough (DvH). In these mutants, the activity of Sat has been artificially increased by perturbing the (i) transcriptional repressor Rex and (ii) its binding site upstream of the gene encoding Sat (Christensen et al., 2015 J. Bacteriol). It was predicted that this would minimize the back reaction of Sat, enhance the intracellular pool of APS, and minimize the equilibration between sulfite and adenosine monophosphate (AMP). Both mutants, along with the wild type DvH were grown in batch culture made with water enriched in 18O. Samples were collected throughout batch growth, and we report the evolution of the S and O isotopic composition of sulfate, and of the S isotopic

  1. Heme-assisted S-Nitrosation Desensitizes Ferric Soluble Guanylate Cyclase to Nitric Oxide*

    PubMed Central

    Fernhoff, Nathaniel B.; Derbyshire, Emily R.; Underbakke, Eric S.; Marletta, Michael A.

    2012-01-01

    Nitric oxide (NO) signaling regulates key processes in cardiovascular physiology, specifically vasodilation, platelet aggregation, and leukocyte rolling. Soluble guanylate cyclase (sGC), the mammalian NO sensor, transduces an NO signal into the classical second messenger cyclic GMP (cGMP). NO binds to the ferrous (Fe2+) oxidation state of the sGC heme cofactor and stimulates formation of cGMP several hundred-fold. Oxidation of the sGC heme to the ferric (Fe3+) state desensitizes the enzyme to NO. The heme-oxidized state of sGC has emerged as a potential therapeutic target in the treatment of cardiovascular disease. Here, we investigate the molecular mechanism of NO desensitization and find that sGC undergoes a reductive nitrosylation reaction that is coupled to the S-nitrosation of sGC cysteines. We further characterize the kinetics of NO desensitization and find that heme-assisted nitrosothiol formation of β1Cys-78 and β1Cys-122 causes the NO desensitization of ferric sGC. Finally, we provide evidence that the mechanism of reductive nitrosylation is gated by a conformational change of the protein. These results yield insights into the function and dysfunction of sGC in cardiovascular disease. PMID:23093402

  2. DgcA, a diguanylate cyclase from Xanthomonas oryzae pv. oryzae regulates bacterial pathogenicity on rice

    PubMed Central

    Su, Jianmei; Zou, Xia; Huang, Liangbo; Bai, Tenglong; Liu, Shu; Yuan, Meng; Chou, Shan-Ho; He, Ya-Wen; Wang, Haihong; He, Jin

    2016-01-01

    Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice blight disease as well as a serious phytopathogen worldwide. It is also one of the model organisms for studying bacteria-plant interactions. Current progress in bacterial signal transduction pathways has identified cyclic di-GMP as a major second messenger molecule in controlling Xanthomonas pathogenicity. However, it still remains largely unclear how c-di-GMP regulates the secretion of bacterial virulence factors in Xoo. In this study, we focused on the important roles played by DgcA (XOO3988), one of our previously identified diguanylate cyclases in Xoo, through further investigating the phenotypes of several dgcA-related mutants, namely, the dgcA-knockout mutant ΔdgcA, the dgcA overexpression strain OdgcA, the dgcA complemented strain CdgcA and the wild-type strain. The results showed that dgcA negatively affected virulence, EPS production, bacterial autoaggregation and motility, but positively triggered biofilm formation via modulating the intracellular c-di-GMP levels. RNA-seq data further identified 349 differentially expressed genes controlled by DgcA, providing a foundation for a more solid understanding of the signal transduction pathways in Xoo. Collectively, the present study highlights DgcA as a major regulator of Xoo virulence, and can serve as a potential target for preventing rice blight diseases. PMID:27193392

  3. Enzymatic Production of c-di-GMP Using a Thermophilic Diguanylate Cyclase.

    PubMed

    Venkataramani, Prabhadevi; Liang, Zhao-Xun

    2017-01-01

    C-di-GMP has emerged as a prevalent bacterial messenger that controls a multitude of bacterial behaviors. Having access to milligram or gram quantities of c-di-GMP is essential for the biochemical and structural characterization of enzymes and effectors involved in c-di-GMP signaling. Although c-di-GMP can be synthesized using chemical methods, diguanylate cyclases (DGC)-based enzymatic synthesis is the most efficient method of preparing c-di-GMP today. Many DGCs are not suitable for c-di-GMP production because of poor protein stability and the presence of a c-di-GMP-binding inhibitory site (I-site) in most DGCs. We have identified and engineered a thermophilic DGC for efficient production of c-di-GMP for characterizing c-di-GMP signaling proteins and riboswitches. Importantly, residue replacement in the inhibitory I-site of the thermophilic DGC drastically relieved product inhibition to enable the production of hundreds of milligrams of c-di-GMP using 5-10 mg of this robust biocatalyst.

  4. Targeting transcriptional control of soluble guanylyl cyclase via NOTCH for prevention of cardiovascular disease.

    PubMed

    Rippe, Catarina; Albinsson, Sebastian; Guron, Gregor; Nilsson, Holger; Swärd, Karl

    2018-05-13

    Soluble guanylyl cyclase (sGC) is an effector enzyme of nitric oxide (NO). Recent work has unraveled how levels of this enzyme are controlled, and highlighted a role in vascular disease. We provide a timely summary of available knowledge on transcriptional regulation of sGC, including influences from the NOTCH signaling pathway and genetic variants. It is speculated that hypertension-induced repression of sGC starts a vicious circle that can be initiated by periods of stress, diet or genetic factors, and a key tenet is that reduction of sGC further raises blood pressure. The idea that dysregulation of sGC contributes to syndromes caused by defective NOTCH signaling is advanced, and we discuss drug repositioning for vascular disease prevention. The advantage of targeting sGC expression rather than activity is also considered. It is argued that transcriptional inputs on sGC arise from interactions with other cells, the extracellular matrix, and microRNAs (miRNAs), and concluded that the promise of sGC as a target for prevention of cardiovascular disease has increased in recent time. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  5. Bioinformatics analysis of the oxidosqualene cyclase gene and the amino acid sequence in mangrove plants

    NASA Astrophysics Data System (ADS)

    Basyuni, M.; Wati, R.

    2017-01-01

    This study described the bioinformatics methods to analyze seven oxidosqualene cyclase (OSC) genes from mangrove plants on DDBJ/EMBL/GenBank as well as predicted the structure, composition, similarity, subcellular localization and phylogenetic. The physical and chemical properties of seven mangrove OSC showed variation among the genes. The percentage of the secondary structure of seven mangrove OSC genes followed the order of a helix > random coil > extended chain structure. The values of chloroplast or signal peptide were too low, indicated that no chloroplast transit peptide or signal peptide of secretion pathway in mangrove OSC genes. The target peptide value of mitochondria varied from 0.163 to 0.430, indicated it was possible to exist. These results suggested the importance of understanding the diversity and functional of properties of the different amino acids in mangrove OSC genes. To clarify the relationship among the mangrove OSC gene, a phylogenetic tree was constructed. The phylogenetic tree shows that there are three clusters, Kandelia KcMS join with Bruguiera BgLUS, Rhizophora RsM1 was close to Bruguiera BgbAS, and Rhizophora RcCAS join with Kandelia KcCAS. The present study, therefore, supported the previous results that plant OSC genes form distinct clusters in the tree.

  6. Identification of a fourth family of lycopene cyclases in photosynthetic bacteria

    PubMed Central

    Maresca, Julia A.; Graham, Joel E.; Wu, Martin; Eisen, Jonathan A.; Bryant, Donald A.

    2007-01-01

    A fourth and large family of lycopene cyclases was identified in photosynthetic prokaryotes. The first member of this family, encoded by the cruA gene of the green sulfur bacterium Chlorobium tepidum, was identified in a complementation assay with a lycopene-producing strain of Escherichia coli. Orthologs of cruA are found in all available green sulfur bacterial genomes and in all cyanobacterial genomes that lack genes encoding CrtL- or CrtY-type lycopene cyclases. The cyanobacterium Synechococcus sp. PCC 7002 has two homologs of CruA, denoted CruA and CruP, and both were shown to have lycopene cyclase activity. Although all characterized lycopene cyclases in plants are CrtL-type proteins, genes orthologous to cruP also occur in plant genomes. The CruA- and CruP-type carotenoid cyclases are members of the FixC dehydrogenase superfamily and are distantly related to CrtL- and CrtY-type lycopene cyclases. Identification of these cyclases fills a major gap in the carotenoid biosynthetic pathways of green sulfur bacteria and cyanobacteria. PMID:17606904

  7. Identification of a fourth family of lycopene cyclases in photosynthetic bacteria.

    PubMed

    Maresca, Julia A; Graham, Joel E; Wu, Martin; Eisen, Jonathan A; Bryant, Donald A

    2007-07-10

    A fourth and large family of lycopene cyclases was identified in photosynthetic prokaryotes. The first member of this family, encoded by the cruA gene of the green sulfur bacterium Chlorobium tepidum, was identified in a complementation assay with a lycopene-producing strain of Escherichia coli. Orthologs of cruA are found in all available green sulfur bacterial genomes and in all cyanobacterial genomes that lack genes encoding CrtL- or CrtY-type lycopene cyclases. The cyanobacterium Synechococcus sp. PCC 7002 has two homologs of CruA, denoted CruA and CruP, and both were shown to have lycopene cyclase activity. Although all characterized lycopene cyclases in plants are CrtL-type proteins, genes orthologous to cruP also occur in plant genomes. The CruA- and CruP-type carotenoid cyclases are members of the FixC dehydrogenase superfamily and are distantly related to CrtL- and CrtY-type lycopene cyclases. Identification of these cyclases fills a major gap in the carotenoid biosynthetic pathways of green sulfur bacteria and cyanobacteria.

  8. The Effects of Thrombin on Adenyl Cyclase Activity and a Membrane Protein from Human Platelets

    PubMed Central

    Brodie, G. N.; Baenziger, Nancy Lewis; Chase, Lewis R.; Majerus, Philip W.

    1972-01-01

    Washed human platelets were incubated with 0.1-1.0 U/ml human thrombin and the effects on adenyl cyclase activity and on a platelet membrane protein (designated thrombin-sensitive protein) were studied. Adenyl cyclase activity was decreased 70-90% when intact platelets were incubated with thrombin. The T½ for loss of adenyl cyclase activity was less than 15 sec at 1 U/ml thrombin. There was no decrease of adenyl cyclase activity when sonicated platelets or isolated membranes were incubated with these concentrations of thrombin. Loss of adenyl cyclase activity was relatively specific since the activities of other platelet membrane enzymes were unaffected by thrombin. Prior incubation of platelets with dibutyryl cyclic adenosine monophosphate (AMP), prostaglandin E1, or theophylline protected adenyl cyclase from inhibition by thrombin. Incubation of intact but not disrupted platelets with thrombin resulted in the release of thrombin-sensitive protein from the platelet membrane. The rapid release of this protein (T½ < 15 sec) at low concentrations of thrombin suggested that removal of thrombin-sensitive protein from the platelet membrane is an integral part of the platelet release reaction. This hypothesis is supported by the parallel effects of thrombin on adenyl cyclase activity and thrombin-sensitive protein release in the presence of dibutyryl cyclic AMP, prostaglandin E1, and theophylline at varying concentrations of thrombin. Images PMID:4331802

  9. Comparison of the effects of histamine and tolazoline on adenylate cyclase activity from guinea pig heart.

    PubMed

    Weinryb, I; Michel, I M

    1975-01-01

    Both histamine and tolazoline (2-benzyl-2-imidazoline) stimulated particulate fractions of adenylate cyclase from guinea pig myocardium. Tolazoline was one-tenth as potent, and about two-thirds as active at maximally effective levels, as was histamine. Enhancement of cyclase activity by tolazoline was additive with that by isoproterenol, and the histamine and tolazoline concentration-response curves were parallel, suggesting that tolazoline acted at the same site as histamine. At maximally effective concentrations, tolazoline did not affect ATPase or cyclic AMP phosphodiesterase activities associated with the cyclase preparations. The H1-receptor antagonist mepyramine, and the H2 antagonist, burimamide, blocked stimulation of cyclase by tolazoline at one-tenth the molarity of agonist. Both antagonists were less effective vs. histamine stimulation of heart cyclase in particulate fractions or whole homogenates, with mepyramine being generally more potent. It is suggested that the molecular basis of the stimulatory effect of tolazoline on cardiac tissue may be histaminergic stimulation of adenylate cyclase. Furthermore, the lack of potency of burimamide as a histamine antagonist and its lack of specificity compared to mepyramine, at the subcellular level, indicate that histamine-responsive adenylate cyclase from heart may not be a satisfactory molecular model for the H2 receptor pharmacology of histamine in cardiac tissue.

  10. Compartmentalized cAMP Signaling Associated With Lipid Raft and Non-raft Membrane Domains in Adult Ventricular Myocytes.

    PubMed

    Agarwal, Shailesh R; Gratwohl, Jackson; Cozad, Mia; Yang, Pei-Chi; Clancy, Colleen E; Harvey, Robert D

    2018-01-01

    Aim: Confining cAMP production to discrete subcellular locations makes it possible for this ubiquitous second messenger to elicit unique functional responses. Yet, factors that determine how and where the production of this diffusible signaling molecule occurs are incompletely understood. The fluid mosaic model originally proposed that signal transduction occurs through random interactions between proteins diffusing freely throughout the plasma membrane. However, it is now known that the movement of membrane proteins is restricted, suggesting that the plasma membrane is segregated into distinct microdomains where different signaling proteins can be concentrated. In this study, we examined what role lipid raft and non-raft membrane domains play in compartmentation of cAMP signaling in adult ventricular myocytes. Methods and Results: The freely diffusible fluorescence resonance energy transfer-based biosensor Epac2-camps was used to measure global cytosolic cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. We found that β-adrenergic receptors, which are expressed in lipid raft and non-raft domains, produce cAMP responses near the plasma membrane that are distinctly different from those produced by E-type prostaglandin receptors, which are expressed exclusively in non-raft domains. We also found that there are differences in basal cAMP levels associated with lipid raft and non-raft domains, and that this can be explained by differences in basal adenylyl cyclase activity associated with each of these membrane environments. In addition, we found evidence that phosphodiesterases 2, 3, and 4 work together in regulating cAMP activity associated with both lipid raft and non-raft domains, while phosphodiesterase 3 plays a more prominent role in the bulk cytoplasmic compartment. Conclusion: These results suggest that different membrane

  11. Downregulated expression of the cyclase-associated protein 1 (CAP1) reduces migration in esophageal squamous cell carcinoma.

    PubMed

    Li, Mei; Yang, Xiaojing; Shi, Hui; Ren, Hanru; Chen, Xueyu; Zhang, Shu; Zhu, Junya; Zhang, Jianguo

    2013-09-01

    Overexpression of cyclase-associated proteins has been associated with poor prognosis in several human cancers. Cyclase-associated protein 1 is a member of the cyclase-associated proteins which contributes to tumor progression. The aim of the present study was to examine the expression of cyclase-associated protein 1 and to elucidate its clinicopathologic significance in a larger series of esophageal squamous cell carcinoma. Immunohistochemical and western blot analyses were performed in esophageal squamous cell carcinoma tissues. Survival analyses were performed by using the Kaplan-Meier method. The role of cyclase-associated protein 1 in migration was studied in esophageal squamous cell carcinoma cell lines of TE1 through knocking down cyclase-associated protein 1 with siRNA and overexpression of cyclase-associated protein 1. The regulation of cyclase-associated protein 1 on migration was determined by transwell and wound-healing assays. Immunohistochemical analysis showed that cyclase-associated protein 1 expression was negatively associated with E-cadherin and significantly associated with lymph node metastases. Survival analysis revealed that cyclase-associated protein 1 overexpression was significantly associated with overall survival (P = 0.011). Knock down of cyclase-associated protein 1 in TE1 cells resulted in decreased vimentin and F-actin levels and the capability for migration. In addition, overexpression of cyclase-associated protein 1 promoted the migration of TE1 cells. These findings suggest that cyclase-associated protein 1 is involved in the metastasis of esophageal squamous cell carcinoma, and that elevated levels of cyclase-associated protein 1 expression may indicate a poor prognosis for patients with esophageal squamous cell carcinoma.

  12. Cardiovascular and adenylate cyclase stimulating effects of colforsin daropate, a water-soluble forskolin derivative, compared with those of isoproterenol, dopamine and dobutamine.

    PubMed

    Yoneyama, Masahiko; Sugiyama, Atsushi; Satoh, Yoshioki; Takahara, Akira; Nakamura, Yuji; Hashimoto, Keitaro

    2002-12-01

    Colforsin daropate is a recently developed water-soluble derivative of forskolin that directly stimulates adenylate cyclase, unlike the catecholamines. The chronotropic, inotropic and coronary vasodilator actions of colforsin daropate were compared with those of isoproterenol, dopamine and dobutamine, using canine isolated, blood-perfused heart preparations. The stimulating effect of each drug on adenylate cyclase activity was also assessed. Colforsin daropate, as well as each of the catecholamines, exerted positive chronotropic, inotropic and coronary vasodilator actions. The order of selectivity for the cardiovascular variables of colforsin daropate was coronary vasodilation > positive inotropy > positive chronotropy; whereas that of isoproterenol, dopamine and dobutamine was positive inotropy > coronary vasodilation > positive chronotropy. Thus, a marked characteristic of colforsin daropate is its potent coronary vasodilator action. On the other hand, each drug significantly increased the adenylate cyclase activity in a dose-related manner: colforsin daropate > isoproterenol > dopamine = dobutamine. These results suggest that colforsin daropate may be preferable in the treatment of severe heart failure where the coronary blood flow is reduced and beta-adrenoceptor-dependent signal transduction pathway is down-regulated.

  13. Prokaryotic adenylate cyclase toxin stimulates anterior pituitary cells in culture

    SciTech Connect

    Cronin, M.J.; Evans, W.S.; Rogol, A.D.

    1986-08-01

    Bordetella pertussis synthesis a variety of virulence factors including a calmodulin-dependent adenylate cyclase (AC) toxin. Treatment of anterior pituitary cells with this AC toxin resulted in an increase in cellular cAMP levels that was associated with accelerated exocytosis of growth hormone (GH), prolactin, adrenocorticotropic hormone (ACTH), and luteinizing hormone (LH). The kinetics of release of these hormones, however, were markedly different; GH and prolactin were rapidly released, while LH and ACTH secretion was more gradually elevated. Neither dopamine agonists nor somatostatin changes the ability of AC toxin to generate cAMP (up to 2 h). Low concentrations of AC toxin amplifiedmore » the secretory response to hypophysiotrophic hormones. The authors conclude that bacterial AC toxin can rapidly elevate cAMP levels in anterior pituitary cells and that it is the response that explains the subsequent acceleration of hormone release.« less

  14. Solubilization and other studies on adenylate cyclase of baker's yeast.

    PubMed Central

    Varimo, K; Londesborough, J

    1976-01-01

    1. Adenylate cyclase of Saccharomyces cerevisiae was sedimented from mechanically disintegrated preparations of yeast over an unusually wide range of centrifugal forces. 2. The enzyme was readily solubilized by Ficoll and by Lubrol PX. Lubrol caused a 2-fold activation. 3. Both particle-bound and Lubrol-solubilized enzyme had an apparent Km for ATP of 1.6 mM in the presence of 0.4 mM-cyclic AMP and 5 mM-MnCl2 at pH 6.2 and 30 degrees C. 4. The Lubrol-solubilized enzyme behaved on gel filtration as a monodisperse protein with an apparent mol.wt. of about 450000. PMID:793584

  15. The temperature-dependence of adenylate cyclase from baker's yeast.

    PubMed Central

    Londesborough, J; Varimo, K

    1979-01-01

    The Michaelis constant of membrane-bound adenylate cyclase increased from 1.1 to 1.8 mM between 7 and 38 degrees C (delta H = 13 kJ/mol). Over this temperature range, the maximum velocity increased 10-fold, and the Arrhenius plot was nearly linear, with an average delta H* of 51 kJ/mol. The temperature-dependence of the reaction rate at 2 mM-ATP was examined in more detail: for Lubrol-dispersed enzyme, Arrhenius plots were nearly linear with average delta H* values of 45 and 68 kJ/mol, respectively, for untreated and gel-filtered enzymes; for membrane-bound enzyme, delta H changed from 40 kJ/mol above about 21 degrees C to 62 kJ/mol below 21 degrees C, but this behaviour does not necessarily indicate an abrupt, lipid-induced, transition in the reaction mechanism. PMID:391221

  16. The cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells

    SciTech Connect

    Cho, Eun-Ah; Juhnn, Yong-Sung, E-mail: juhnn@snu.ac.kr

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer cAMP signaling system inhibits repair of {gamma}-ray-induced DNA damage. Black-Right-Pointing-Pointer cAMP signaling system inhibits DNA damage repair by decreasing XRCC1 expression. Black-Right-Pointing-Pointer cAMP signaling system decreases XRCC1 expression by promoting its proteasomal degradation. Black-Right-Pointing-Pointer The promotion of XRCC1 degradation by cAMP signaling system is mediated by Epac1. -- Abstract: Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNAmore » repair activity, and we investigated the effects of the cAMP signaling system on {gamma}-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (G{alpha}sQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of G{alpha}sQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after {gamma}-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2 Prime -O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2 Prime -O-Me-cAMP and restored XRCC1 protein level following {gamma

  17. Identification of a soluble guanylate cyclase in RBCs: preserved activity in patients with coronary artery disease.

    PubMed

    Cortese-Krott, Miriam M; Mergia, Evanthia; Kramer, Christian M; Lückstädt, Wiebke; Yang, Jiangning; Wolff, Georg; Panknin, Christina; Bracht, Thilo; Sitek, Barbara; Pernow, John; Stasch, Johannes-Peter; Feelisch, Martin; Koesling, Doris; Kelm, Malte

    2018-04-01

    Endothelial dysfunction is associated with decreased NO bioavailability and impaired activation of the NO receptor soluble guanylate cyclase (sGC) in the vasculature and in platelets. Red blood cells (RBCs) are known to produce NO under hypoxic and normoxic conditions; however evidence of expression and/or activity of sGC and downstream signaling pathway including phopshodiesterase (PDE)-5 and protein kinase G (PKG) in RBCs is still controversial. In the present study, we aimed to investigate whether RBCs carry a functional sGC signaling pathway and to address whether this pathway is compromised in coronary artery disease (CAD). Using two independent chromatographic procedures, we here demonstrate that human and murine RBCs carry a catalytically active α 1 β 1 -sGC (isoform 1), which converts 32 P-GTP into 32 P-cGMP, as well as PDE5 and PKG. Specific sGC stimulation by NO+BAY 41-2272 increases intracellular cGMP-levels up to 1000-fold with concomitant activation of the canonical PKG/VASP-signaling pathway. This response to NO is blunted in α1-sGC knockout (KO) RBCs, but fully preserved in α2-sGC KO. In patients with stable CAD and endothelial dysfunction red cell eNOS expression is decreased as compared to aged-matched controls; by contrast, red cell sGC expression/activity and responsiveness to NO are fully preserved, although sGC oxidation is increased in both groups. Collectively, our data demonstrate that an intact sGC/PDE5/PKG-dependent signaling pathway exists in RBCs, which remains fully responsive to NO and sGC stimulators/activators in patients with endothelial dysfunction. Targeting this pathway may be helpful in diseases with NO deficiency in the microcirculation like sickle cell anemia, pulmonary hypertension, and heart failure. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  18. Hydrogen peroxide stimulation of CFTR reveals an Epac-mediated, soluble AC-dependent cAMP amplification pathway common to GPCR signalling

    PubMed Central

    Ivonnet, P; Salathe, M; Conner, G E

    2015-01-01

    BACKGROUND AND PURPOSE H2O2 is widely understood to regulate intracellular signalling. In airway epithelia, H2O2 stimulates anion secretion primarily by activating an autocrine PGE2 signalling pathway via EP4 and EP1 receptors to initiate cytic fibrosis transmembrane regulator (CFTR)-mediated Cl− secretion. This study investigated signalling downstream of the receptors activated by H2O2. EXPERIMENTAL APPROACH Anion secretion by differentiated bronchial epithelial cells was measured in Ussing chambers during stimulation with H2O2, an EP4 receptor agonist or β2-adrenoceptor agonist in the presence and absence of inhibitors of ACs and downstream effectors. Intracellular calcium ([Ca2+]I) changes were followed by microscopy using fura–2-loaded cells and PKA activation followed by FRET microscopy. KEY RESULTS Transmembrane adenylyl cyclase (tmAC) and soluble AC (sAC) were both necessary for H2O2 and EP4 receptor-mediated CFTR activation in bronchial epithelia. H2O2 and EP4 receptor agonist stimulated tmAC to increase exchange protein activated by cAMP (Epac) activity that drives PLC activation to raise [Ca2+]i via Ca2+ store release (and not entry). Increased [Ca2+]i led to sAC activation and further increases in CFTR activity. Stimulation of sAC did not depend on changes in [HCO3−]. Ca2+-activated apical KCa1.1 channels and cAMP-activated basolateral KV7.1 channels contributed to H2O2-stimulated anion currents. A similar Epac-mediated pathway was seen following β2-adrenoceptor or forskolin stimulation. CONCLUSIONS AND IMPLICATIONS H2O2 initiated a complex signalling cascade that used direct stimulation of tmACs by Gαs followed by Epac-mediated Ca2+ crosstalk to activate sAC. The Epac-mediated Ca2+ signal constituted a positive feedback loop that amplified CFTR anion secretion following stimulation of tmAC by a variety of stimuli. PMID:25220136

  19. Dual contradictory roles of cAMP signaling pathways in hydroxyl radical production in the rat striatum.

    PubMed

    Hara, Shuichi; Kobayashi, Masamune; Kuriiwa, Fumi; Mukai, Toshiji; Mizukami, Hajime

    2012-03-15

    Studies have suggested that cAMP signaling pathways may be associated with the production of reactive oxygen species. In this study, we examined how modifications in cAMP signaling affected the production of hydroxyl radicals in rat striatum using microdialysis to measure extracellular 2,3-dihydroxybenzoic acid (2,3-DHBA), which is a hydroxyl radical adduct of salicylate. Up to 50 nmol of the cell-permeative cAMP mimetic 8-bromo-cAMP (8-Br-cAMP) increased 2,3-DHBA in a dose-dependent manner (there was no additional increase in 2,3-DHBA at 100 nmol). Another cAMP mimetic, dibutyryl cAMP (db-cAMP), caused a nonsignificant increase in 2,3-DHBA at 50 nmol and a significant decrease at 100 nmol. Up to 20 nmol of forskolin, which is a direct activator of adenylyl cyclase, increased 2,3-DHBA, similar to the effect of 8-Br-cAMP; however, forskolin resulted in a much greater increase in 2,3-DHBA. A potent inhibitor of protein kinase A (PKA), H89 (500 μM), potentiated the 8-Br-cAMP- and forskolin-induced increases in 2,3-DHBA and antagonized the inhibitory effect of 100 nmol of db-cAMP. Interestingly, the administration of 100 nmol of 8-bromo-cGMP alone or in combination with H89 had no significant effect on 2,3-DHBA levels. Doses of 100 nmol of a preferential PKA activator (6-phenyl-cAMP) or a preferential PKA inhibitor (8-bromoadenosine-3',5'-cyclic monophosphorothionate, Rp-isomer; Rp-8-Br-cAMPS), which also inhibits the cAMP-mediated activation of Epac (the exchange protein directly activated by cAMP), suppressed or enhanced, respectively, the formation of 2,3-DHBA. Up to 100 nmol of 8-(4-chlorophenylthio)-2'-O-methyladenosine-cAMP, which is a selective activator of Epac, dose-dependently stimulated the formation of 2,3-DHBA. These findings suggest that cAMP signaling plays contradictory roles (stimulation and inhibition) in the production of hydroxyl radicals in rat striatum by differential actions of Epac and PKA. These roles might contribute to the production of

  20. Opioid-receptor (OR) signaling cascades in rat cerebral cortex and model cell lines: the role of plasma membrane structure.

    PubMed

    Ujčíková, H; Brejchová, J; Vošahlíková, M; Kagan, D; Dlouhá, K; Sýkora, J; Merta, L; Drastichová, Z; Novotný, J; Ostašov, P; Roubalová, L; Parenti, M; Hof, M; Svoboda, P

    2014-01-01

    Large number of extracellular signals is received by plasma membrane receptors which, upon activation, transduce information into the target cell interior via trimeric G-proteins (GPCRs) and induce activation or inhibition of adenylyl cyclase enzyme activity (AC). Receptors for opioid drugs such as morphine (micro-OR, delta-OR and kappa-OR) belong to rhodopsin family of GPCRs. Our recent results indicated a specific up-regulation of AC I (8-fold) and AC II (2.5-fold) in plasma membranes (PM) isolated from rat brain cortex exposed to increasing doses of morphine (10-50 mg/kg) for 10 days. Increase of ACI and ACII represented the specific effect as the amount of ACIII-ACIX, prototypical PM marker Na, K-ATPase and trimeric G-protein alpha and beta subunits was unchanged. The up-regulation of ACI and ACII faded away after 20 days since the last dose of morphine. Proteomic analysis of these PM indicated that the brain cortex of morphine-treated animals cannot be regarded as being adapted to this drug because significant up-regulation of proteins functionally related to oxidative stress and alteration of brain energy metabolism occurred. The number of delta-OR was increased 2-fold and their sensitivity to monovalent cations was altered. Characterization of delta-OR-G-protein coupling in model HEK293 cell line indicated high ability of lithium to support affinity of delta-OR response to agonist stimulation. Our studies of PM structure and function in context with desensitization of GPCRs action were extended by data indicating participation of cholesterol-enriched membrane domains in agonist-specific internalization of delta-OR. In HEK293 cells stably expressing delta-OR-G(i)1alpha fusion protein, depletion of PM cholesterol was associated with the decrease in affinity of G-protein response to agonist stimulation, whereas maximum response was unchanged. Hydrophobic interior of isolated PM became more "fluid", chaotically organized and accessible to water molecules

  1. Interactions between lysergic acid diethylamide and dopamine-sensitive adenylate cyclase systems in rat brain.

    PubMed

    Hungen, K V; Roberts, S; Hill, D F

    1975-08-22

    Investigations were carried out on the interactions of the hallucinogenic drug, D-lysergic acid diethylamide (D-LSD), and other serotonin antagonists with catecholamine-sensitive adenylate cyclase systems in cell-free preparations from different regions of rat brain. In equimolar concentration, D-LSD, 2-brono-D-lysergic acid diethylamide (BOL), or methysergide (UML) strongly blocked maximal stimulation of adenylate cyclase activity by either norepinephrine or dopamine in particulate preparations from cerebral cortices of young adult rats. D-LSD also eliminated the stimulation of adenylate cyclase activity of equimolar concentrations of norepinephrine or dopamine in particulate preparations from rat hippocampus. The effects of this hallucinogenic agent on adenylate cyclase activity were most striking in particulate preparations from corpus striatum. Thus, in 10 muM concentration, D-LSD not only completely eradicated the response to 10 muM dopamine in these preparations but also consistently stimulated adenylate cyclase activity. L-LSD (80 muM) was without effect. Significant activation of striatal adenylate cyclase was produced by 0.1 muM D-LSD. Activation of striatal adenylate cyclase of either D-LSD or dopamine was strongly blocked by the dopamine-blocking agents trifluoperazine, thioridazine, chlorpromazine, and haloperidol. The stimulatory effects of D-LSD and dopamine were also inhibited by the serotonin-blocking agents, BOL, 1-methyl-D-lysergic acid diethylamide (MLD), and cyproheptadine, but not by the beta-adrenergic-blocking agent, propranolol. However, these serotonin antagonists by themselves were incapable of stimulating adenylate cyclase activity in the striatal preparations. Several other hallucinogens, which were structurally related to serotonin, were also inactive in this regard, e.g., mescaline, N,N-dimethyltryptamine, psilocin and bufotenine. Serotonin itself produced a small stimulation of adenylate cyclase activity in striatal preparations and

  2. A nitric oxide/cysteine interaction mediates the activation of soluble guanylate cyclase

    PubMed Central

    Fernhoff, Nathaniel B.; Derbyshire, Emily R.; Marletta, Michael A.

    2009-01-01

    Nitric oxide (NO) regulates a number of essential physiological processes by activating soluble guanylate cyclase (sGC) to produce the second messenger cGMP. The mechanism of NO sensing was previously thought to result exclusively from NO binding to the sGC heme; however, recent studies indicate that heme-bound NO only partially activates sGC and additional NO is involved in the mechanism of maximal NO activation. Furthermore, thiol oxidation of sGC cysteines results in the loss of enzyme activity. Herein the role of cysteines in NO-stimulated sGC activity investigated. We find that the thiol modifying reagent methyl methanethiosulfonate specifically inhibits NO activation of sGC by blocking a non-heme site, which defines a role for sGC cysteine(s) in mediating NO binding. The nature of the NO/cysteine interaction was probed by examining the effects of redox active reagents on NO-stimulated activity. These results show that NO binding to, and dissociation from, the critical cysteine(s) does not involve a change in the thiol redox state. Evidence is provided for non-heme NO in the physiological activation of sGC in context of a primary cell culture of human umbilical vein endothelial cells. These findings have relevance to diseases involving the NO/cGMP signaling pathway. PMID:20007374

  3. Accelerated Evolution of the Pituitary Adenylate Cyclase-Activating Polypeptide Precursor Gene During Human Origin

    PubMed Central

    Wang, Yin-qiu; Qian, Ya-ping; Yang, Su; Shi, Hong; Liao, Cheng-hong; Zheng, Hong-Kun; Wang, Jun; Lin, Alice A.; Cavalli-Sforza, L. Luca; Underhill, Peter A.; Chakraborty, Ranajit; Jin, Li; Su, Bing

    2005-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide abundantly expressed in the central nervous system and involved in regulating neurogenesis and neuronal signal transduction. The amino acid sequence of PACAP is extremely conserved across vertebrate species, indicating a strong functional constraint during the course of evolution. However, through comparative sequence analysis, we demonstrated that the PACAP precursor gene underwent an accelerated evolution in the human lineage since the divergence from chimpanzees, and the amino acid substitution rate in humans is at least seven times faster than that in other mammal species resulting from strong Darwinian positive selection. Eleven human-specific amino acid changes were identified in the PACAP precursors, which are conserved from murine to African apes. Protein structural analysis suggested that a putative novel neuropeptide might have originated during human evolution and functioned in the human brain. Our data suggested that the PACAP precursor gene underwent adaptive changes during human origin and may have contributed to the formation of human cognition. PMID:15834139

  4. Glomerular Podocytes Express Type 1 Adenylate Cyclase: Inactivation Results in Susceptibility to Proteinuria

    PubMed Central

    Xiao, Zhijie; He, Liqun; Takemoto, Minoru; Jalanko, Hannu; Chan, Guy C.; Storm, Daniel R.; Betsholtz, Christer; Tryggvason, Karl; Patrakka, Jaakko

    2011-01-01

    Background/Aims The organization of actin cytoskeleton in podocyte foot processes plays a critical role in the maintenance of the glomerular filtration barrier. The cAMP pathway is an important regulator of the actin network assembly in cells. However, the role of the cAMP pathway in podocytes is not well understood. Type 1 adenylate cyclase (Adcy1), previously thought to be specific for neuronal tissue, is a member of the family of enzymes that catalyses the formation of cAMP. In this study, we characterized the expression and role of Adcy1 in the kidney. Methods Expression of Adcy1 was studied by RT-PCR, Northern blotting and in situ hybridization. The role of Adcy1 in podocytes was investigated by analyzing Adcy1 knockout mice (Adcy1–/–). Results and Conclusion: Adcy1 is expressed in the kidney specifically by podocytes. In the kidney, Adcy1 does not have a critical role in normal physiological functioning as kidney histology and function are normal in Adcy1–/– mice. However, albumin overload resulted in severe albuminuria in Adcy1–/– mice, whereas wild-type control mice showed only mild albumin leakage to urine. In conclusion, we have identified Adcy1 as a novel podocyte signaling protein that seems to have a role in compensatory physiological processes in the glomerulus. PMID:21196775

  5. Dimerization Domain of Retinal Membrane Guanylyl Cyclase 1 (RetGC1) Is an Essential Part of Guanylyl Cyclase-activating Protein (GCAP) Binding Interface.

    PubMed

    Peshenko, Igor V; Olshevskaya, Elena V; Dizhoor, Alexander M

    2015-08-07

    The photoreceptor-specific proteins guanylyl cyclase-activating proteins (GCAPs) bind and regulate retinal membrane guanylyl cyclase 1 (RetGC1) but not natriuretic peptide receptor A (NPRA). Study of RetGC1 regulation in vitro and its association with fluorescently tagged GCAP in transfected cells showed that R822P substitution in the cyclase dimerization domain causing congenital early onset blindness disrupted RetGC1 ability to bind GCAP but did not eliminate its affinity for another photoreceptor-specific protein, retinal degeneration 3 (RD3). Likewise, the presence of the NPRA dimerization domain in RetGC1/NPRA chimera specifically disabled binding of GCAPs but not of RD3. In subsequent mapping using hybrid dimerization domains in RetGC1/NPRA chimera, multiple RetGC1-specific residues contributed to GCAP binding by the cyclase, but the region around Met(823) was the most crucial. Either positively or negatively charged residues in that position completely blocked GCAP1 and GCAP2 but not RD3 binding similarly to the disease-causing mutation in the neighboring Arg(822). The specificity of GCAP binding imparted by RetGC1 dimerization domain was not directly related to promoting dimerization of the cyclase. The probability of coiled coil dimer formation computed for RetGC1/NPRA chimeras, even those incapable of binding GCAP, remained high, and functional complementation tests showed that the RetGC1 active site, which requires dimerization of the cyclase, was formed even when Met(823) or Arg(822) was mutated. These results directly demonstrate that the interface for GCAP binding on RetGC1 requires not only the kinase homology region but also directly involves the dimerization domain and especially its portion containing Arg(822) and Met(823). © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Effect of nitroso complexes of some transition metals on the activity of soluble guanylate cyclase.

    PubMed

    Severina, I S; Bussygina, O G; Grigorjev, N B

    1992-03-01

    Effects of nitroso complexes of some transition metals (Fe, Co, Cr), differing in the character of NO oxidation on the activity of human and rat platelet guanylate cyclase were studied. 3 types of nitroso complexes were used: (1) NO group carries a positive charge--a nitrosonium cation (Na2[FeNO + (CN)5]-nitroprusside); (2) NO is neutral--(K3[CrNO(CN)5 and [CoNO(NH3)5]SO4) and (3) NO is coordinated as anion NO- (K3[CoNO-(CN)5]. It is shown that the highest stimulatory effect is produced by sodium nitroprusside, whose activating action is due to the interaction of its NO group with the guanylate cyclase heme. Nitroso complexes (Co and Cr) the NO group of which is neutral stimulated guanylate cyclase activity insignificantly and this activation was not guanylate cyclase heme directed. Nitroso complex (Co) with NO coordinated as anion NO(-)--is a guanylate cyclase inhibitor. In contrast to nitroprusside, the nitroso complexes used (Co and Cr) have no hypotensive effect. It was concluded that the essential requirement for the realization of the hypotensive effect of transition metals' nitroso complexes is the ability of these compounds to activate soluble guanylate cyclase solely by the heme-dependent mechanism.

  7. Pituitary adenylate cyclase-activating polypeptide: a novel peptide with protean implications.

    PubMed

    Pisegna, Joseph R; Oh, David S

    2007-02-01

    The purpose of this review is to highlight the importance of pituitary adenylate cyclase-activating polypeptide in physiological processes and to describe how this peptide is becoming increasingly recognized as having a major role in the body. Since its discovery in 1989, investigators have sought to determine the site of biological activity and the function of pituitary adenylate cyclase-activating polypeptide in maintaining homeostasis. Since its discovery, pituitary adenylate cyclase-activating polypeptide appears to play an important role in the regulation of processes within the central nervous system and gastrointestinal tract, as well in reproductive biology. Pituitary adenylate cyclase-activating polypeptide has been shown to regulate tumor cell growth and to regulate immune function through its effects on T lympocytes. These discoveries suggest the importance of pituitary adenylate cyclase-activating polypeptide in neuronal development, neuronal function, gastrointestinal tract function and reproduction. Future studies will examine more closely the role of pituitary adenylate cyclase-activating polypeptide in regulation of malignantly transformed cells, as well as in regulation of immune function.

  8. Cholera toxin activation of adenylate cyclase in cancer cell membrane fragments.

    PubMed Central

    Bitensky, M W; Wheeler, M A; Mehta, H; Miki, N

    1975-01-01

    Activation of adenylate [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by cholera toxin (84,000 daltons, 5.5 S) is demonstrated in plasma membrane fragments of mouse ascites cancer cells. The activation of adenylate cyclase is mediated by a macromolecular cyclase activating factor (MCAF), which has a sedimentation constant of 2.7 S and a molecular weight of about 26,000. MCAF is derived from, and may be identical to the "A fragment" of cholera toxin. Generation of MCAF depends on prior interaction of cholera toxin with either dithiothreitol, NADH, NAD, or a low-molecular-weight component (less than 700 daltons) present in cytoplasm. Subsequent exposure of this pretreated cholera toxin to cell membranes from a variety of mouse ascites cancer cells is followed rapidly by the appearance of MCAF, which no longer requires dithiothreitol, NADH, or NAD for the activation of adenylate cyclase. Activation of adenylate cyclase by MCAF in ascites cancer cell membrane fragments is not reversed by repeated washing of these membrane fragments. Adenylate cyclase in normal cell membrane fragments fails to respond either to cholera toxin or MCAF in the presence of dithiothreitol. In striking contrast, the adenylate cyclase in membrane fragments from five ascites cancer cells responds to either MCAF or native cholera toxin preincubated with dithiothreitol, NADH, or NAD. PMID:1058474

  9. Action of Escherichia coli Enterotoxin: Adenylate Cyclase Behavior of Intestinal Epithelial Cells in Culture

    PubMed Central

    Kantor, Harvey S.; Tao, Pearl; Wisdom, Charlene

    1974-01-01

    Heat-labile enterotoxin preparations obtained from two enteropathogenic strains of Escherichia coli of porcine and human origin were shown to stimulate adenylate cyclase activity of human embryonic intestinal epithelial cells in culture. Comparable results were also obtained when cholera toxin was used. The degree of enzyme stimulation was proportional to the concentration of enterotoxin. Similar preparations from two strains of non-enterotoxigenic E. coli had no effect on adenylate cyclase activity. Cells exposed to enterotoxin could be washed after 1 min of contact time without altering the subsequent course of maximum adenylate cyclase activity, which was maintained for at least 18 h at 37 C. During long periods (18 h) of tissue culture incubation, the determination of adenylate cyclase activity was 200- to 300-fold more sensitive than quantitating fluid accumulation in the adult rabbit ileal loop model. Decreasing the incubation time appreciably reduced the sensitivity of the epithelial cells to enterotoxin. E. coli enterotoxin is an effective activator of nonintestinal adenylate cyclase systems. Treatment of KB and HEp-2 cell lines with enterotoxin also resulted in significant enzyme stimulation. The intestinal epithelial cell tissue culture model provides a sensitive homogenous biological system for studying the response of intestinal adenylate cyclase to enterotoxin while eliminating the numerous cellular and tissue components present in the ligated ileal loop model. PMID:4364505

  10. The Cyclase-Associated Protein Cap1 Is Important for Proper Regulation of Infection-Related Morphogenesis in Magnaporthe oryzae

    PubMed Central

    Zhou, Xiaoying; Zhang, Haifeng; Li, Guotian; Shaw, Brian; Xu, Jin-Rong

    2012-01-01

    Surface recognition and penetration are critical steps in the infection cycle of many plant pathogenic fungi. In Magnaporthe oryzae, cAMP signaling is involved in surface recognition and pathogenesis. Deletion of the MAC1 adenylate cyclase gene affected appressorium formation and plant infection. In this study, we used the affinity purification approach to identify proteins that are associated with Mac1 in vivo. One of the Mac1-interacting proteins is the adenylate cyclase-associated protein named Cap1. CAP genes are well-conserved in phytopathogenic fungi but none of them have been functionally characterized. Deletion of CAP1 blocked the effects of a dominant RAS2 allele and resulted in defects in invasive growth and a reduced intracellular cAMP level. The Δcap1 mutant was defective in germ tube growth, appressorium formation, and formation of typical blast lesions. Cap1-GFP had an actin-like localization pattern, localizing to the apical regions in vegetative hyphae, at the periphery of developing appressoria, and in circular structures at the base of mature appressoria. Interestingly, Cap1, similar to LifeAct, did not localize to the apical regions in invasive hyphae, suggesting that the apical actin cytoskeleton differs between vegetative and invasive hyphae. Domain deletion analysis indicated that the proline-rich region P2 but not the actin-binding domain (AB) of Cap1 was responsible for its subcellular localization. Nevertheless, the AB domain of Cap1 must be important for its function because CAP1 ΔAB only partially rescued the Δcap1 mutant. Furthermore, exogenous cAMP induced the formation of appressorium-like structures in non-germinated conidia in CAP1 ΔAB transformants. This novel observation suggested that AB domain deletion may result in overstimulation of appressorium formation by cAMP treatment. Overall, our results indicated that CAP1 is important for the activation of adenylate cyclase, appressorium morphogenesis, and plant infection in M

  11. Cyclic Nucleotide-Gated Channels, Calmodulin, Adenylyl Cyclase, and Calcium/Calmodulin-Dependent Protein Kinase II Are Required for Late, but Not Early, Long-Term Memory Formation in the Honeybee

    ERIC Educational Resources Information Center

    Matsumoto, Yukihisa; Sandoz, Jean-Christophe; Devaud, Jean-Marc; Lormant, Flore; Mizunami, Makoto; Giurfa, Martin

    2014-01-01

    Memory is a dynamic process that allows encoding, storage, and retrieval of information acquired through individual experience. In the honeybee "Apis mellifera," olfactory conditioning of the proboscis extension response (PER) has shown that besides short-term memory (STM) and mid-term memory (MTM), two phases of long-term memory (LTM)…

  12. Roles of Protein Kinase A and Adenylate Cyclase in Light-Modulated Cellulase Regulation in Trichoderma reesei

    PubMed Central

    Schuster, André; Tisch, Doris; Seidl-Seiboth, Verena; Kubicek, Christian P.

    2012-01-01

    The cyclic AMP (cAMP) pathway represents a central signaling cascade with crucial functions in all organisms. Previous studies of Trichoderma reesei (anamorph of Hypocrea jecorina) suggested a function of cAMP signaling in regulation of cellulase gene expression. We were therefore interested in how the crucial components of this pathway, adenylate cyclase (ACY1) and cAMP-dependent protein kinase A (PKA), would affect cellulase gene expression. We found that both ACY1 and PKA catalytic subunit 1 (PKAC1) are involved in regulation of vegetative growth but are not essential for sexual development. Interestingly, our results showed considerably increased transcript abundance of cellulase genes in darkness compared to light (light responsiveness) upon growth on lactose. This effect is strongly enhanced in mutant strains lacking PKAC1 or ACY1. Comparison to the wild type showed that ACY1 has a consistently positive effect on cellulase gene expression in light and darkness, while PKAC1 influences transcript levels of cellulase genes positively in light but negatively in darkness. A function of PKAC1 in light-modulated cellulase gene regulation is also reflected by altered complex formation within the cel6a/cbh2 promoter in light and darkness and in the absence of pkac1. Analysis of transcript levels of cellulase regulator genes indicates that the regulatory output of the cAMP pathway may be established via adjustment of XYR1 abundance. Consequently, both adenylate cyclase and protein kinase A are involved in light-modulated cellulase gene expression in T. reesei and have a dampening effect on the light responsiveness of this process. PMID:22286997

  13. Thrombospondin-1 and Angiotensin II Inhibit Soluble Guanylyl Cyclase through an Increase in Intracellular Calcium Concentration

    PubMed Central

    Ramanathan, Saumya; Mazzalupo, Stacy; Boitano, Scott; Montfort, William R.

    2011-01-01

    Nitric Oxide (NO) regulates cardiovascular hemostasis by binding to soluble guanylyl cyclase (sGC), leading to cGMP production, reduced cytosolic calcium concentration ([Ca2+]i) and vasorelaxation. Thrombospondin-1 (TSP-1), a secreted matricellular protein, was recently discovered to inhibit NO signaling and sGC activity. Inhibition of sGC requires binding to cell-surface receptor CD47. Here, we show that a TSP-1 C-terminal fragment (E3CaG1) readily inhibits sGC in Jurkat T cells, and that inhibition requires an increase in [Ca2+]i. Using flow cytometry, we show that E3CaG1 binds directly to CD47 on the surface of Jurkat T cells. Using digital imaging microscopy on live cells, we further show that E3CaG1 binding results in a substantial increase in [Ca2+]i, up to 300 nM. Addition of angiotensin II, a potent vasoconstrictor known to increase [Ca2+]i, also strongly inhibits sGC activity. sGC isolated from calcium-treated cells or from cell-free lysates supplemented with Ca2+ remains inhibited, while addition of kinase inhibitor staurosporine prevents inhibition, indicating inhibition is likely due to phosphorylation. Inhibition is through an increase in Km for GTP, which rises to 834 µM for the NO-stimulated protein, a 13-fold increase over the uninhibited protein. Compounds YC-1 and BAY 41-2272, allosteric stimulators of sGC that are of interest for treating hypertension, overcome E3CaG1-mediated inhibition of NO-ligated sGC. Taken together, these data suggest that sGC not only lowers [Ca2+]i in response to NO, inducing vasodilation, but is also inhibited by high [Ca2+]i, providing a fine balance between signals for vasodilation and vasoconstriction. PMID:21823650

  14. Synthesis of arborane triterpenols by a bacterial oxidosqualene cyclase

    NASA Astrophysics Data System (ADS)

    Banta, Amy B.; Wei, Jeremy H.; Gill, Clare C. C.; Giner, José-Luis; Welander, Paula V.

    2017-01-01

    Cyclic triterpenoids are a broad class of polycyclic lipids produced by bacteria and eukaryotes. They are biologically relevant for their roles in cellular physiology, including membrane structure and function, and biochemically relevant for their exquisite enzymatic cyclization mechanism. Cyclic triterpenoids are also geobiologically significant as they are readily preserved in sediments and are used as biomarkers for ancient life throughout Earth's history. Isoarborinol is one such triterpenoid whose only known biological sources are certain angiosperms and whose diagenetic derivatives (arboranes) are often used as indicators of terrestrial input into aquatic environments. However, the occurrence of arborane biomarkers in Permian and Triassic sediments, which predates the accepted origin of angiosperms, suggests that microbial sources of these lipids may also exist. In this study, we identify two isoarborinol-like lipids, eudoraenol and adriaticol, produced by the aerobic marine heterotrophic bacterium Eudoraea adriatica. Phylogenetic analysis demonstrates that the E. adriatica eudoraenol synthase is an oxidosqualene cyclase homologous to bacterial lanosterol synthases and distinct from plant triterpenoid synthases. Using an Escherichia coli heterologous sterol expression system, we demonstrate that substitution of four amino acid residues in a bacterial lanosterol synthase enabled synthesis of pentacyclic arborinols in addition to tetracyclic sterols. This variant provides valuable mechanistic insight into triterpenoid synthesis and reveals diagnostic amino acid residues to differentiate between sterol and arborinol synthases in genomic and metagenomic datasets. Our data suggest that there may be additional bacterial arborinol producers in marine and freshwater environments that could expand our understanding of these geologically informative lipids.

  15. Human recombinant soluble guanylyl cyclase: expression, purification, and regulation

    NASA Technical Reports Server (NTRS)

    Lee, Y. C.; Martin, E.; Murad, F.

    2000-01-01

    The alpha1- and beta1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells/baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3-(5'-hydroxymethyl-2'furyl)-1-benzyl-indazole (YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other in combination and demonstrated a remarkable 2,200-fold stimulation of the human recombinant sGC. The effects were inhibited with 1H-(1,2, 4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the recombinant enzyme with respect to GTP was examined. The products of the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent of inhibition by cGMP depended on the activation state of the enzyme, whereas inhibition by pyrophosphate was not affected by the enzyme state. Both reaction products displayed independent binding and cooperativity with respect to enzyme inhibition. The expression of large quantities of active enzyme will facilitate structural characterization of the protein.

  16. Bicarbonate Modulates Photoreceptor Guanylate Cyclase (ROS-GC) Catalytic Activity*

    PubMed Central

    Duda, Teresa; Wen, Xiao-Hong; Isayama, Tomoki; Sharma, Rameshwar K.; Makino, Clint L.

    2015-01-01

    By generating the second messenger cGMP in retinal rods and cones, ROS-GC plays a central role in visual transduction. Guanylate cyclase-activating proteins (GCAPs) link cGMP synthesis to the light-induced fall in [Ca2+]i to help set absolute sensitivity and assure prompt recovery of the response to light. The present report discloses a surprising feature of this system: ROS-GC is a sensor of bicarbonate. Recombinant ROS-GCs synthesized cGMP from GTP at faster rates in the presence of bicarbonate with an ED50 of 27 mm for ROS-GC1 and 39 mm for ROS-GC2. The effect required neither Ca2+ nor use of the GCAPs domains; however, stimulation of ROS-GC1 was more powerful in the presence of GCAP1 or GCAP2 at low [Ca2+]. When applied to retinal photoreceptors, bicarbonate enhanced the circulating current, decreased sensitivity to flashes, and accelerated flash response kinetics. Bicarbonate was effective when applied either to the outer or inner segment of red-sensitive cones. In contrast, bicarbonate exerted an effect when applied to the inner segment of rods but had little efficacy when applied to the outer segment. The findings define a new regulatory mechanism of the ROS-GC system that affects visual transduction and is likely to affect the course of retinal diseases caused by cGMP toxicity. PMID:25767116

  17. Synthesis of arborane triterpenols by a bacterial oxidosqualene cyclase

    PubMed Central

    Banta, Amy B.; Wei, Jeremy H.; Gill, Clare C. C.; Giner, José-Luis; Welander, Paula V.

    2017-01-01

    Cyclic triterpenoids are a broad class of polycyclic lipids produced by bacteria and eukaryotes. They are biologically relevant for their roles in cellular physiology, including membrane structure and function, and biochemically relevant for their exquisite enzymatic cyclization mechanism. Cyclic triterpenoids are also geobiologically significant as they are readily preserved in sediments and are used as biomarkers for ancient life throughout Earth's history. Isoarborinol is one such triterpenoid whose only known biological sources are certain angiosperms and whose diagenetic derivatives (arboranes) are often used as indicators of terrestrial input into aquatic environments. However, the occurrence of arborane biomarkers in Permian and Triassic sediments, which predates the accepted origin of angiosperms, suggests that microbial sources of these lipids may also exist. In this study, we identify two isoarborinol-like lipids, eudoraenol and adriaticol, produced by the aerobic marine heterotrophic bacterium Eudoraea adriatica. Phylogenetic analysis demonstrates that the E. adriatica eudoraenol synthase is an oxidosqualene cyclase homologous to bacterial lanosterol synthases and distinct from plant triterpenoid synthases. Using an Escherichia coli heterologous sterol expression system, we demonstrate that substitution of four amino acid residues in a bacterial lanosterol synthase enabled synthesis of pentacyclic arborinols in addition to tetracyclic sterols. This variant provides valuable mechanistic insight into triterpenoid synthesis and reveals diagnostic amino acid residues to differentiate between sterol and arborinol synthases in genomic and metagenomic datasets. Our data suggest that there may be additional bacterial arborinol producers in marine and freshwater environments that could expand our understanding of these geologically informative lipids. PMID:28028245

  18. A novel antithrombotic effect of sulforaphane via activation of platelet adenylate cyclase: ex vivo and in vivo studies.

    PubMed

    Jayakumar, Thanasekaran; Chen, Wei-Fan; Lu, Wan-Jung; Chou, Duen-Suey; Hsiao, George; Hsu, Chung-Yi; Sheu, Joen-Rong; Hsieh, Cheng-Ying

    2013-06-01

    study demonstrates for the first time that in addition to it originally being considered as an agent for prevention of tumor growth, sulforaphane possesses potent antiplatelet activity which may initially activate adenylate cyclase/cAMP, followed by inhibiting intracellular signals (such as the PI3-kinase/Akt and PLCγ2-PKC-p47 cascades) and ultimately inhibiting platelet activation. Therefore, this novel role of sulforaphane may represent a high therapeutic potential for treatment or prevention of cardiovascular diseases. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. High-throughput screening using the differential radial capillary action of ligand assay identifies ebselen as an inhibitor of diguanylate cyclases.

    PubMed

    Lieberman, Ori J; Orr, Mona W; Wang, Yan; Lee, Vincent T

    2014-01-17

    The rise of bacterial resistance to traditional antibiotics has motivated recent efforts to identify new drug candidates that target virulence factors or their regulatory pathways. One such antivirulence target is the cyclic-di-GMP (cdiGMP) signaling pathway, which regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Mutations that abrogate cdiGMP binding to these receptors prevent polysaccharide production. Identification of small molecules that can inhibit cdiGMP binding to the allosteric sites on these proteins could mimic binding defective mutants and potentially reduce biofilm formation or alginate secretion. Here, we report the development of a rapid and quantitative high-throughput screen for inhibitors of protein-cdiGMP interactions based on the differential radial capillary action of ligand assay (DRaCALA). Using this approach, we identified ebselen as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in P. aeruginosa through inhibition of diguanylate cyclases. The identification of ebselen provides a proof-of-principle that a DRaCALA high-throughput screening approach can be used to identify bioactive agents that reverse regulation of cdiGMP signaling by targeting cdiGMP-binding domains.

  20. Factors affecting the activity of guanylate cyclase in lysates of human blood platelets.

    PubMed Central

    Adams, A F; Haslam, R J

    1978-01-01

    1. Under optimal ionic conditions (4 mM-MnCl2) the specific activity of guanylate cyclase in fresh platelet lysates was about 10nmol of cyclic GMP formed/20 min per mg of protein at 30 degrees C. Activity was 15% of optimum with 10mM-MgCl2 and negligible with 4mM-CaCl2. Synergism between MnCl2 and MgCl2 or CaCl2 was observed when [MnCl2] less than or equal to [GPT]. 2. Lower than optimal specific activities were obtained in assays containing large volumes of platelet lysate, owing to the presence of inhibitory factors that could be removed by ultrafiltration. Adenine nucleotides accounted for less than 50% of the inhibitory activity. 3. Preincubation of lysate for 1 h at 30 degrees C increased the specific activity of platelet guanylate cyclase by about 2-fold. 4. Lubrol PX (1%, w/v) stimulated guanylate cyclase activity by 3--5-fold before preincubation and by about 2-fold after preincubation. Triton X-100 was much less effective. 5. Dithiothreitol inhibited the guanylate cyclase activity of untreated, preincubated and Lubrol PX-treated lysates and prevented activation by preincubation provided that it was added beforehand. 6. Oleate stimulated guanylate cyclase activity 3--4-fold and arachidonate 2--3-fold, whereas palmitate was almost inactive. Pretreatment of lysate with indomethacin did not inhibit this effect of arachidonate. Oleate and arachidonate caused marked stimulation of guanylate cyclase in preincubated lysate, but inhibited the enzyme in Lubrol PX-treated lysate. 7. NaN3 (10mM) increased guanylate cyclase activity by up to 7-fold; this effect was both time- and temperature-dependent. NaN3 did not further activate the enzyme in Lubrol PX-treated lysate. 8. The results indicated that preincubation, Lubrol PX, fatty acids and NaN3 activated platelet guanylate cyclase by different mechanisms. 9. Platelet particulate fractions contained no guanylate cyclase activity detectable in the presence or absence of Lubrol PX that could not be accounted for by

  1. Factors affecting the activity of guanylate cyclase in lysates of human blood platelets.

    PubMed

    Adams, A F; Haslam, R J

    1978-07-15

    1. Under optimal ionic conditions (4 mM-MnCl2) the specific activity of guanylate cyclase in fresh platelet lysates was about 10nmol of cyclic GMP formed/20 min per mg of protein at 30 degrees C. Activity was 15% of optimum with 10mM-MgCl2 and negligible with 4mM-CaCl2. Synergism between MnCl2 and MgCl2 or CaCl2 was observed when [MnCl2] less than or equal to [GPT]. 2. Lower than optimal specific activities were obtained in assays containing large volumes of platelet lysate, owing to the presence of inhibitory factors that could be removed by ultrafiltration. Adenine nucleotides accounted for less than 50% of the inhibitory activity. 3. Preincubation of lysate for 1 h at 30 degrees C increased the specific activity of platelet guanylate cyclase by about 2-fold. 4. Lubrol PX (1%, w/v) stimulated guanylate cyclase activity by 3--5-fold before preincubation and by about 2-fold after preincubation. Triton X-100 was much less effective. 5. Dithiothreitol inhibited the guanylate cyclase activity of untreated, preincubated and Lubrol PX-treated lysates and prevented activation by preincubation provided that it was added beforehand. 6. Oleate stimulated guanylate cyclase activity 3--4-fold and arachidonate 2--3-fold, whereas palmitate was almost inactive. Pretreatment of lysate with indomethacin did not inhibit this effect of arachidonate. Oleate and arachidonate caused marked stimulation of guanylate cyclase in preincubated lysate, but inhibited the enzyme in Lubrol PX-treated lysate. 7. NaN3 (10mM) increased guanylate cyclase activity by up to 7-fold; this effect was both time- and temperature-dependent. NaN3 did not further activate the enzyme in Lubrol PX-treated lysate. 8. The results indicated that preincubation, Lubrol PX, fatty acids and NaN3 activated platelet guanylate cyclase by different mechanisms. 9. Platelet particulate fractions contained no guanylate cyclase activity detectable in the presence or absence of Lubrol PX that could not be accounted for by

  2. Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides.

    PubMed

    Gagne, Steve J; Stout, Jake M; Liu, Enwu; Boubakir, Zakia; Clark, Shawn M; Page, Jonathan E

    2012-07-31

    Δ(9)-Tetrahydrocannabinol (THC) and other cannabinoids are responsible for the psychoactive and medicinal properties of Cannabis sativa L. (marijuana). The first intermediate in the cannabinoid biosynthetic pathway is proposed to be olivetolic acid (OA), an alkylresorcinolic acid that forms the polyketide nucleus of the cannabinoids. OA has been postulated to be synthesized by a type III polyketide synthase (PKS) enzyme, but so far type III PKSs from cannabis have been shown to produce catalytic byproducts instead of OA. We analyzed the transcriptome of glandular trichomes from female cannabis flowers, which are the primary site of cannabinoid biosynthesis, and searched for polyketide cyclase-like enzymes that could assist in OA cyclization. Here, we show that a type III PKS (tetraketide synthase) from cannabis trichomes requires the presence of a polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which catalyzes a C2-C7 intramolecular aldol condensation with carboxylate retention to form OA. OAC is a dimeric α+β barrel (DABB) protein that is structurally similar to polyketide cyclases from Streptomyces species. OAC transcript is present at high levels in glandular trichomes, an expression profile that parallels other cannabinoid pathway enzymes. Our identification of OAC both clarifies the cannabinoid pathway and demonstrates unexpected evolutionary parallels between polyketide biosynthesis in plants and bacteria. In addition, the widespread occurrence of DABB proteins in plants suggests that polyketide cyclases may play an overlooked role in generating plant chemical diversity.

  3. LH-RH binding to purified pituitary plasma membranes: absence of adenylate cyclase activation.

    PubMed

    Clayton, R N; Shakespear, R A; Marshall, J C

    1978-06-01

    Purified bovine pituitary plasma membranes possess two specific LH-RH binding sites. The high affinity site (2.5 X 10(9) l/mol) has low capacity (9 X 10(-15) mol/mg membrane protein) while the low affinity site 6.1 X 10(5) l/mol) has a much higher capacity (1.1 X 10(-10) mol/mg). Specific LH-RH binding to plasma membranes is increased 8.5-fold during purification from homogenate whilst adenylate cyclase activity is enriched 7--8-fold. Distribution of specific LH-RH binding to sucrose density gradient interface fractions parallels that of adenylate cyclase activity. Mg2+ and Ca2+ inhibit specific [125I]LH-RH binding at micromolar concentrations. Synthetic LH-RH, up to 250 microgram/ml, failed to stimulate adenylase cyclase activity of the purified bovine membranes. Using a crude 10,800 g rat pituitary membrane preparation, LH-RH similarly failed to activate adenylate cyclase even in the presence of guanyl nucleotides. These data confirm the presence of LH-RH receptor sites on pituitary plasma membranes and suggest that LH-RH-induced gonadotrophin release may be mediated by mechanisms other than activation of adenylate cyclase.

  4. Nitric oxide (NO), the only nitrogen monoxide redox form capable of activating soluble guanylyl cyclase.

    PubMed

    Dierks, E A; Burstyn, J N

    1996-06-28

    In the present study, we determined that of the redox forms of nitrogen monoxide, NO-, NO and NO+, only NO significantly activates soluble guanylyl cyclase (GTP pyrophosphate-lyase cyclizing, EC 4.6.1.2). Neither of the NO-donors tested, Angeli's salt (Na2N2O3) or Piloty's acid (C6H5SO2NHOH), caused a change in the guanylyl cyclase activity relative to the basal activity level. Interference by other reaction products was eliminated as a possible explanation for the lack of activation. To the extent that NO+ could be stabilized in aqueous solution, by dissolution of the nitrosonium salt NOPF6 in dry organic solvent prior to addition to the enzyme in buffer, NO+ had no effect on the activity of soluble guanylyl cyclase. The counter-ion, PF6-, had a minimal effect on the enzyme activity and, therefore was, not responsible for the lack of activation by NO+. These observations suggest that NO- is the natural activator of soluble guanylyl cyclase and is reasonably identical with endothelium-derived relaxing factor, the physiological regulator of soluble guanylyl cyclase activity.

  5. Effect of age and posture on human lymphocyte adenylate cyclase activity.

    PubMed

    Mader, S L; Robbins, A S; Rubenstein, L Z; Tuck, M L; Scarpace, P J

    1988-03-01

    1. A number of age-related changes have been reported in the catecholamine-adrenoceptor-adenylate cyclase system. Most of the data available on these alterations come from resting subjects; the response to acute stress may provide additional insights into the age effect on these responses. 2. We measured supine and 10 min upright plasma noradrenaline and lymphocyte adenylate cyclase activity in ten healthy elderly subjects (age 66-80 years) and seven healthy young subjects (age 27-34 years). 3. Isoprenaline stimulation of lymphocyte adenylate cyclase activity was not significantly different between supine and upright positions or between elderly and young subjects. There was a marked increase in forskolin-stimulated adenylate cyclase activity in the upright posture in both elderly and young subjects. The increment over supine levels was 70% in the elderly (P less than 0.025) and 73% in the young (P less than 0.05). This enhanced forskolin activity was not seen in two young subjects who became syncopal. 4. These data suggest that enhanced forskolin-stimulated adenylate cyclase activity occurs after 10 min of upright posture in both elderly and young subjects, and may be relevant to immediate blood pressure regulation. We were unable to demonstrate any age-related differences in these acute adrenergic responses.

  6. Cloning and Functional Characterization of a Lycopene β-Cyclase from Macrophytic Red Alga Bangia fuscopurpurea.

    PubMed

    Cao, Tian-Jun; Huang, Xing-Qi; Qu, Yuan-Yuan; Zhuang, Zhong; Deng, Yin-Yin; Lu, Shan

    2017-04-11

    Lycopene cyclases cyclize the open ends of acyclic lycopene (ψ,ψ-carotene) into β- or ε-ionone rings in the crucial bifurcation step of carotenoid biosynthesis. Among all carotenoid constituents, β-carotene (β,β-carotene) is found in all photosynthetic organisms, except for purple bacteria and heliobacteria, suggesting a ubiquitous distribution of lycopene β-cyclase activity in these organisms. In this work, we isolated a gene ( BfLCYB ) encoding a lycopene β-cyclase from Bangia fuscopurpurea , a red alga that is considered to be one of the primitive multicellular eukaryotic photosynthetic organisms and accumulates carotenoid constituents with both β- and ε-rings, including β-carotene, zeaxanthin, α-carotene (β,ε-carotene) and lutein. Functional complementation in Escherichia coli demonstrated that BfLCYB is able to catalyze cyclization of lycopene into monocyclic γ-carotene (β,ψ-carotene) and bicyclic β-carotene, and cyclization of the open end of monocyclic δ-carotene (ε,ψ-carotene) to produce α-carotene. No ε-cyclization activity was identified for BfLCYB. Sequence comparison showed that BfLCYB shares conserved domains with other functionally characterized lycopene cyclases from different organisms and belongs to a group of ancient lycopene cyclases. Although B. fuscopurpurea also synthesizes α-carotene and lutein, its enzyme-catalyzing ε-cyclization is still unknown.

  7. Pore-formation by adenylate cyclase toxoid activates dendritic cells to prime CD8+ and CD4+ T cells.

    PubMed

    Svedova, Martina; Masin, Jiri; Fiser, Radovan; Cerny, Ondrej; Tomala, Jakub; Freudenberg, Marina; Tuckova, Ludmila; Kovar, Marek; Dadaglio, Gilles; Adkins, Irena; Sebo, Peter

    2016-04-01

    The adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis is a bi-functional leukotoxin. It penetrates myeloid phagocytes expressing the complement receptor 3 and delivers into their cytosol its N-terminal adenylate cyclase enzyme domain (~400 residues). In parallel, ~1300 residue-long RTX hemolysin moiety of CyaA forms cation-selective pores and permeabilizes target cell membrane for efflux of cytosolic potassium ions. The non-enzymatic CyaA-AC(-) toxoid, has repeatedly been successfully exploited as an antigen delivery tool for stimulation of adaptive T-cell immune responses. We show that the pore-forming activity confers on the CyaA-AC(-) toxoid a capacity to trigger Toll-like receptor and inflammasome signaling-independent maturation of CD11b-expressing dendritic cells (DC). The DC maturation-inducing potency of mutant toxoid variants in vitro reflected their specifically enhanced or reduced pore-forming activity and K(+) efflux. The toxoid-induced in vitro phenotypic maturation of DC involved the activity of mitogen activated protein kinases p38 and JNK and comprised increased expression of maturation markers, interleukin 6, chemokines KC and LIX and granulocyte-colony-stimulating factor secretion, prostaglandin E2 production and enhancement of chemotactic migration of DC. Moreover, i.v. injected toxoids induced maturation of splenic DC in function of their cell-permeabilizing capacity. Similarly, the capacity of DC to stimulate CD8(+) and CD4(+) T-cell responses in vitro and in vivo was dependent on the pore-forming activity of CyaA-AC(-). This reveals a novel self-adjuvanting capacity of the CyaA-AC(-) toxoid that is currently under clinical evaluation as a tool for delivery of immunotherapeutic anti-cancer CD8(+) T-cell vaccines into DC.

  8. Enzymatic synthesis and characterizations of cyclic GDP-ribose. A procedure for distinguishing enzymes with ADP-ribosyl cyclase activity.

    PubMed

    Graeff, R M; Walseth, T F; Fryxell, K; Branton, W D; Lee, H C

    1994-12-02

    Cyclic nucleotides such as cAMP and cGMP are second messengers subserving various signaling pathways. Cyclic ADP-ribose (cADPR), a recently discovered member of the family, is derived from NAD+ and is a mediator of Ca2+ mobilization in various cellular systems. The synthesis and degradation of cADPR are, respectively, catalyzed by ADP-ribosyl cyclase and cADPR hydrolase. CD38, a differentiation antigen of B lymphocytes, has recently been shown to be a bifunctional enzyme catalyzing both the formation and hydrolysis of cADPR. The overall reaction catalyzed by CD38 is the formation of ADP-ribose and nicotinamide from NAD+, identical to that catalyzed by NADase. The difficulties in detecting the formation of cADPR have led to frequent identification of CD38 as a classical NADase. In this study, we show that both ADP-ribosyl cyclase and CD38, but not NADase, can cyclize nicotinamide guanine dinucleotide (NGD+) producing a new nucleotide. Analyses by high performance liquid chromatography and mass spectroscopy indicate the product is cyclic GDP-ribose (cGDPR) with a structure similar to cADPR except with guanine replacing adenine. Compared to cADPR, cGDPR is a more stable compound showing 2.8 times more resistance to heat-induced hydrolysis. These results are consistent with a catalytic scheme for CD38 where the cyclization of the substrate precedes the hydrolytic reaction. Spectroscopic analyses show that cGDPR is fluorescent and has an absorption spectrum different from both NGD+ and GDPR, providing a very convenient way for monitoring its enzymatic formation. The use of NGD+ as substrate for assaying the cyclization reaction was found to be applicable to pure enzymes as well as crude tissue extracts making it a useful diagnostic tool for distinguishing CD38-like enzymes from degradative NADases.

  9. Cyclase inhibitor tripropylamine significantly enhanced lycopene accumulation in Blakeslea trispora.

    PubMed

    Wang, Yanlong; Chen, Xiwen; Hong, Xiao; Du, Shipeng; Liu, Chunxiao; Gong, Wenfang; Chen, Defu

    2016-11-01

    Lycopene is a member of carotenoids that exhibits strong antioxidant activity. In this study, on the basis of screening suitable strain combination [ATCC 14271(+) and ATCC 14272(-)] and establishing the optimal inoculation proportion of mated culture (1/2, +/-, w/w) for carotenoid production, the efficiency of compounds, mainly tertiary amines, on enhancing the lycopene content of Blakeslea trispora was systematically assessed. Of these compounds, tripropylamine showed the best enhancing effect, and then sequentially followed by triethylamine, tributylamine, trimethylamine, diisopropylamine, and isopropylamine. After treated with 1.8 g/L tripropylamine for two days, the lycopene proportion was increased from 1.7% to 90.1%, while the β-carotene proportion was decreased from 91.1% to 6.4% of the total carotenoids. In this case, the lycopene and total carotenoid contents were increased to 83.2 and 92.4 mg/gDW, which were 315.8- and 5.9-fold of that of the untreated control, respectively; while the growth of mycelia was only decreased at 6.0 g/L tripropylamine. Gene expression analysis showed that all the tested genes, especially genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (hmgr) and isopentenyl pyrophosphate isomerase (ipi) in mevalonate pathway, as well as phytoene desaturase (carB) in carotenoid biosynthesis process were upregulated. Therefore, tripropylamine enhanced lycopene content of B. trispora by inhibiting the cyclase activity, and by upregulating the expression of genes associated with terpenoid biosynthesis. Besides, a possible association between the structure and the lycopene-enhancing capability of these compounds was also discussed. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  10. Bicarbonate Modulates Photoreceptor Guanylate Cyclase (ROS-GC) Catalytic Activity.

    PubMed

    Duda, Teresa; Wen, Xiao-Hong; Isayama, Tomoki; Sharma, Rameshwar K; Makino, Clint L

    2015-04-24

    By generating the second messenger cGMP in retinal rods and cones, ROS-GC plays a central role in visual transduction. Guanylate cyclase-activating proteins (GCAPs) link cGMP synthesis to the light-induced fall in [Ca(2+)]i to help set absolute sensitivity and assure prompt recovery of the response to light. The present report discloses a surprising feature of this system: ROS-GC is a sensor of bicarbonate. Recombinant ROS-GCs synthesized cGMP from GTP at faster rates in the presence of bicarbonate with an ED50 of 27 mM for ROS-GC1 and 39 mM for ROS-GC2. The effect required neither Ca(2+) nor use of the GCAPs domains; however, stimulation of ROS-GC1 was more powerful in the presence of GCAP1 or GCAP2 at low [Ca(2+)]. When applied to retinal photoreceptors, bicarbonate enhanced the circulating current, decreased sensitivity to flashes, and accelerated flash response kinetics. Bicarbonate was effective when applied either to the outer or inner segment of red-sensitive cones. In contrast, bicarbonate exerted an effect when applied to the inner segment of rods but had little efficacy when applied to the outer segment. The findings define a new regulatory mechanism of the ROS-GC system that affects visual transduction and is likely to affect the course of retinal diseases caused by cGMP toxicity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Estradiol rapidly inhibits soluble guanylyl cyclase expression in rat uterus

    NASA Technical Reports Server (NTRS)

    Krumenacker, J. S.; Hyder, S. M.; Murad, F.

    2001-01-01

    Previous reports that investigated the regulation of the NO/soluble guanylyl cyclase (sGC)/cGMP pathway by estrogenic compounds have focused primarily on the levels of NO, NO-producing enzymes, and cGMP in various tissues. In this study, we demonstrate that 17beta-estradiol (E2) regulates the alpha(1) and beta(1) subunits of the NO receptor, sGC, at the mRNA and protein levels in rat uterus. Using real-time quantitative PCR, we found that within 1 h of in vivo E2 administration to rats, sGC mRNA levels begin to diminish. After 3 h, there is a maximal diminution of sGC mRNA expression (sGC alpha(1) 10% and sGC beta(1) 33% of untreated). This effect was blocked by the estrogen receptor antagonist, ICI 182,780, indicating that estrogen receptor is required. The effect of E2 also was observed in vitro with incubations of uterine tissue, indicating that the response does not depend on the secondary release of other hormones or factors from other tissues. Puromycin did not block the effect, suggesting the effects occur because of preexisting factors in uterine tissues and do not require new protein synthesis. Using immunoblot analysis, we found that sGC protein levels also were reduced by E2 over a similar time course as the sGC mRNA. We conclude that sGC plays a vital role in the NO/sGC/cGMP regulatory pathway during conditions of elevated estrogen levels in the rat uterus as a result of the reduction of sGC expression.

  12. Molecular Characterization of Tick Salivary Gland Glutaminyl Cyclase

    PubMed Central

    Adamson, Steven W.; Browning, Rebecca E.; Chao, Chien-Chung; Bateman, Robert C.; Ching, Wei-Mei; Karim, Shahid

    2013-01-01

    Glutaminyl cyclase (QC) catalyzes the cyclization of N-terminal glutamine residues into pyroglutamate. This post-translational modification extends the half-life of peptides and, in some cases, is essential in binding to their cognate receptor. Due to its potential role in the post-translational modification of tick neuropeptides, we report the molecular, biochemical and physiological characterization of salivary gland QC during the prolonged blood-feeding of the black-legged tick (Ixodes scapularis) and the gulf-coast tick (Amblyomma maculatum). QC sequences from I. scapularis and A. maculatum showed a high degree of amino acid identity to each other and other arthropods and residues critical for zinc-binding/catalysis (D159, E202, and H330) or intermediate stabilization (E201, W207, D248, D305, F325, and W329) are conserved. Analysis of QC transcriptional gene expression kinetics depicts an upregulation during the blood-meal of adult female ticks prior to fast feeding phases in both I. scapularis and A. maculatum suggesting a functional link with blood meal uptake. QC enzymatic activity was detected in saliva and extracts of tick salivary glands and midguts. Recombinant QC was shown to be catalytically active. Furthermore, knockdown of QC-transcript by RNA interference resulted in lower enzymatic activity, and small, unviable egg masses in both studied tick species as well as lower engorged tick weights for I. scapularis. These results suggest that the post-translational modification of neurotransmitters and other bioactive peptides by QC is critical to oviposition and potentially other physiological processes. Moreover, these data suggest that tick-specific QC-modified neurotransmitters/hormones or other relevant parts of this system could potentially be used as novel physiological targets for tick control. PMID:23770496

  13. Guanylyl cyclase activation reverses resistive breathing-induced lung injury and inflammation.

    PubMed

    Glynos, Constantinos; Toumpanakis, Dimitris; Loverdos, Konstantinos; Karavana, Vassiliki; Zhou, Zongmin; Magkou, Christina; Dettoraki, Maria; Perlikos, Fotis; Pavlidou, Athanasia; Kotsikoris, Vasilis; Topouzis, Stavros; Theocharis, Stamatios E; Brouckaert, Peter; Giannis, Athanassios; Papapetropoulos, Andreas; Vassilakopoulos, Theodoros

    2015-06-01

    Inspiratory resistive breathing (RB), encountered in obstructive lung diseases, induces lung injury. The soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP) pathway is down-regulated in chronic and acute animal models of RB, such as asthma, chronic obstructive pulmonary disease, and in endotoxin-induced acute lung injury. Our objectives were to: (1) characterize the effects of increased concurrent inspiratory and expiratory resistance in mice via tracheal banding; and (2) investigate the contribution of the sGC/cGMP pathway in RB-induced lung injury. Anesthetized C57BL/6 mice underwent RB achieved by restricting tracheal surface area to 50% (tracheal banding). RB for 24 hours resulted in increased bronchoalveolar lavage fluid cellularity and protein content, marked leukocyte infiltration in the lungs, and perturbed respiratory mechanics (increased tissue resistance and elasticity, shifted static pressure-volume curve right and downwards, decreased static compliance), consistent with the presence of acute lung injury. RB down-regulated sGC expression in the lung. All manifestations of lung injury caused by RB were exacerbated by the administration of the sGC inhibitor, 1H-[1,2,4]oxodiazolo[4,3-]quinoxalin-l-one, or when RB was performed using sGCα1 knockout mice. Conversely, restoration of sGC signaling by prior administration of the sGC activator BAY 58-2667 (Bayer, Leverkusen, Germany) prevented RB-induced lung injury. Strikingly, direct pharmacological activation of sGC with BAY 58-2667 24 hours after RB reversed, within 6 hours, the established lung injury. These findings raise the possibility that pharmacological targeting of the sGC-cGMP axis could be used to ameliorate lung dysfunction in obstructive lung diseases.

  14. Abacavir increases platelet reactivity via competitive inhibition of soluble guanylyl cyclase

    PubMed Central

    Baum, Paul D.; Sullam, Paul M.; Stoddart, Cheryl A.; McCune, Joseph M.

    2011-01-01

    Objective To provide a molecular mechanism that explains the association of the antiretroviral guanosine analogue, abacavir, with an increased risk of myocardial infarction. Design Drug effects were studied with biochemical and cellular assays. Methods Human platelets were incubated with nucleoside analogue drugs ex vivo. Platelet activation stimulated by ADP was studied by measuring surface P-selectin with flow cytometry. Inhibition of purified soluble guanylyl cyclase was quantified using an ELISA to measure cGMP production. Results Pre-incubation of platelets in abacavir significantly increased activation in response to ADP in a time and dose-dependent manner. The active anabolite of abacavir, carbovir triphosphate, competitively inhibited soluble guanylyl cyclase activity with a Ki of 55 μmol/l. Conclusion Abacavir competitively inhibits guanylyl cyclase, leading to platelet hyper-reactivity. This may explain the observed increased risk of myocardial infarction in HIV patients taking abacavir. PMID:21941165

  15. Molecular study of a squalene cyclase homolog gene in Bacillus subtilis

    NASA Astrophysics Data System (ADS)

    Bosak, T.; Pearson, A.; Losick, R.

    2005-12-01

    Polycyclic triterpenoids such as hopanes and steranes are formed by enzymatic cyclization of linear isoprenoid precursors by squalene cyclases and oxidosqualene cyclases. Due to their amazing preservation potential, polycyclic triterpenoids have been used to indicate the source of organic matter in oils and sediments for decades, although many cannot be attributed to known organisms and genes. To bridge the gap between the genomic database and the geochemical record, we are using molecular tools to study the expression, intracellular localization, and products of a squalene cyclase homolog found in Bacillus subtilis, a Gram-positive soil bacterium. We find that the gene is expressed during sporulation and is localized to the spore coat. Our results may help to understand the source of some previously unassigned natural products, and they may also provide clues to the physiological role of triterpenoids in the Bacillales.

  16. Dysfunction of outer segment guanylate cyclase caused by retinal disease related mutations

    PubMed Central

    Zägel, Patrick; Koch, Karl-Wilhelm

    2014-01-01

    Membrane bound guanylate cyclases are expressed in rod and cone cells of the vertebrate retina and mutations in several domains of rod outer segment guanylate cyclase 1 (ROS-GC1 encoded by the gene GUCY2D) correlate with different forms of retinal degenerations. In the present work we investigated the biochemical consequences of three point mutations, one is located in position P575L in the juxtamembrane domain close to the kinase homology domain and two are located in the cyclase catalytic domain at H1019P and P1069R. These mutations correlate with various retinal diseases like autosomal dominant progressive cone degeneration, e.g., Leber Congenital Amaurosis and a juvenile form of retinitis pigmentosa. Wildtype and mutant forms of ROS-GC1 were heterologously expressed in HEK cells, their cellular distribution was investigated and activity profiles in the presence and absence of guanylate cyclase-activating proteins were measured. The mutant P575L was active under all tested conditions, but it displayed a twofold shift in the Ca2+-sensitivity, whereas the mutant P1069R remained inactive despite normal expression levels. The mutation H1019P caused the cyclase to become more labile. The different biochemical consequences of these mutations seem to reflect the different clinical symptoms. The mutation P575L induces a dysregulation of the Ca2+-sensitive cyclase activation profile causing a slow progression of the disease by the distortion of the Ca2+-cGMP homeostasis. In contrast, a strong reduction in cGMP synthesis due to an inactive or structurally unstable ROS-GC1 would trigger more severe forms of retinal diseases. PMID:24616660

  17. Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

    SciTech Connect

    Murayama, T.; Ui, M.

    1985-06-25

    Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased /sup 45/Ca/sup 2 +/ uptake into the cell monolayer, and (f) increased /sup 86/Rb/sup +/ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separatemore » effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca/sup 2 +/ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca/sup 2 +/-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca/sup 2 +/ gating.« less

  18. Dephosphorylation of juxtamembrane serines and threonines of the NPR2 guanylyl cyclase is required for rapid resumption of oocyte meiosis in response to luteinizing hormone

    PubMed Central

    Shuhaibar, Leia C.; Egbert, Jeremy R.; Edmund, Aaron B.; Uliasz, Tracy F.; Dickey, Deborah M.; Yee, Siu-Pok; Potter, Lincoln R.; Jaffe, Laurinda A.

    2016-01-01

    The meiotic cell cycle of mammalian oocytes starts during embryogenesis and then pauses until luteinizing hormone (LH) acts on the granulosa cells of the follicle surrounding the oocyte to restart the cell cycle. An essential event in this process is a decrease in cyclic GMP in the granulosa cells, and part of the cGMP decrease results from dephosphorylation and inactivation of the natriuretic peptide receptor 2 (NPR2) guanylyl cyclase, also known as guanylyl cyclase B. However, it is unknown whether NPR2 dephosphorylation is essential for LH-induced meiotic resumption. Here, we prevented NPR2 dephosphorylation by generating a mouse line in which the seven regulatory serines and threonines of NPR2 were changed to the phosphomimetic amino acid glutamate (Npr2–7E). Npr2–7E/7E follicles failed to show a decrease in enzyme activity in response to LH, and the cGMP decrease was attenuated; correspondingly, LH-induced meiotic resumption was delayed. Meiotic resumption in response to EGF receptor activation was likewise delayed, indicating that NPR2 dephosphorylation is a component of the pathway by which EGF receptor activation mediates LH signaling. We also found that most of the NPR2 protein in the follicle was present in the mural granulosa cells. These findings indicate that NPR2 dephosphorylation in the mural granulosa cells is essential for the normal progression of meiosis in response to LH and EGF receptor activation. In addition, these studies provide the first demonstration that a change in phosphorylation of a transmembrane guanylyl cyclase regulates a physiological process, a mechanism that may also control other developmental events. PMID:26522847

  19. Subcellular distribution and activation by non-ionic detergents of guanylate cyclase in cerebral cortex of rat.

    PubMed

    Deguchi, T; Amano, E; Nakane, M

    1976-11-01

    Non-ionic detergents stimulated particulate guanylate cyclase activity in cerebral cortex of rat 8- to 12-fold while stimulation of soluble enzyme was 1.3- to 2.5-fold. Among various detergents, Lubrol PX was the most effective one. The subcellular distribution of guanylate cyclase activity was examined with or without 0.5% Lubrol PX. Without Lubrol PX two-thirds of the enzyme activity was detected in the soluble fraction. In the presence of Lubrol PX, however, two-thirds of guanylate cyclase activity was recovered in the crude mitochondrial fraction. Further fractionation revealed that most of the particulate guanylate cyclase activity was associated with synaptosomes. The sedimentation characteristic of the particulate guanylate cyclase activity was very close to those of choline acetyltransferase and acetylcholine esterase activities, two synaptosomal enzymes. When the crude mitochondrial fraction was subfractionated after osmotic shock, most of guanylate cyclase activity as assayed in the absence of Lubrol PX was released into the soluble fraction while the rest of the enzyme activity was tightly bound to synaptic membrane fractions. The total guanylate cyclase activity recovered in the synaptosomal soluble fraction was 6 to 7 times higher than that of the starting material. The specific enzyme activity reached more than 1000 pmol per min per mg protein, which was 35-fold higher than that of the starting material. The membrane bound guanylate cyclase activity was markedly stimulated by Lubrol PX. Guanylate cyclase activity in the synaptosomal soluble fraction, in contrast, was suppressed by the addition of Lubrol PX. The observation that most of guanylate cyclase activity was detected in synaptosomes, some of which was tightly bound to the synaptic membrane fraction upon hypoosmotic treatment, is consistent with the concept that cyclic GMP is involved in neural transmission.

  20. Coordinated induction of GST and MRP2 by cAMP in Caco-2 cells: Role of protein kinase A signaling pathway and toxicological relevance

    SciTech Connect

    Arana, Maite Rocío, E-mail: arana@ifise-conicet.gov.ar; Tocchetti, Guillermo Nicolás, E-mail: gtocchetti@live.com.ar; Domizi, Pablo, E-mail: domizi@ibr-conicet.gov.ar

    2015-09-01

    The cAMP pathway is a universal signaling pathway regulating many cellular processes including metabolic routes, growth and differentiation. However, its effects on xenobiotic biotransformation and transport systems are poorly characterized. The effect of cAMP on expression and activity of GST and MRP2 was evaluated in Caco-2 cells, a model of intestinal epithelium. Cells incubated with the cAMP permeable analog dibutyryl cyclic AMP (db-cAMP: 1,10,100 μM) for 48 h exhibited a dose–response increase in GST class α and MRP2 protein expression. Incubation with forskolin, an activator of adenylyl cyclase, confirmed the association between intracellular cAMP and upregulation of MRP2. Consistent withmore » increased expression of GSTα and MRP2, db-cAMP enhanced their activities, as well as cytoprotection against the common substrate 1-chloro-2,4-dinitrobenzene. Pretreatment with protein kinase A (PKA) inhibitors totally abolished upregulation of MRP2 and GSTα induced by db-cAMP. In silico analysis together with experiments consisting of treatment with db-cAMP of Caco-2 cells transfected with a reporter construct containing CRE and AP-1 sites evidenced participation of these sites in MRP2 upregulation. Further studies involving the transcription factors CREB and AP-1 (c-JUN, c-FOS and ATF2) demonstrated increased levels of total c-JUN and phosphorylation of c-JUN and ATF2 by db-cAMP, which were suppressed by a PKA inhibitor. Co-immunoprecipitation and ChIP assay studies demonstrated that db-cAMP increased c-JUN/ATF2 interaction, with further recruitment to the region of the MRP2 promoter containing CRE and AP-1 sites. We conclude that cAMP induces GSTα and MRP2 expression and activity in Caco-2 cells via the PKA pathway, thus regulating detoxification of specific xenobiotics. - Highlights: • cAMP positively modulates the expression and activity of GST and MRP2 in Caco-2 cells. • Such induction resulted in increased cytoprotection against chemical injury.

  1. Comparative analysis of diguanylate cyclase and phosphodiesterase genes in Klebsiella pneumoniae.

    PubMed

    Cruz, Diana P; Huertas, Mónica G; Lozano, Marcela; Zárate, Lina; Zambrano, María Mercedes

    2012-07-09

    Klebsiella pneumoniae can be found in environmental habitats as well as in hospital settings where it is commonly associated with nosocomial infections. One of the factors that contribute to virulence is its capacity to form biofilms on diverse biotic and abiotic surfaces. The second messenger Bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) is a ubiquitous signal in bacteria that controls biofilm formation as well as several other cellular processes. The cellular levels of this messenger are controlled by c-di-GMP synthesis and degradation catalyzed by diguanylate cyclase (DGC) and phophodiesterase (PDE) enzymes, respectively. Many bacteria contain multiple copies of these proteins with diverse organizational structure that highlight the complex regulatory mechanisms of this signaling network. This work was undertaken to identify DGCs and PDEs and analyze the domain structure of these proteins in K. pneumoniae. A search for conserved GGDEF and EAL domains in three sequenced K. pneumoniae genomes showed that there were multiple copies of GGDEF and EAL containing proteins. Both single domain and hybrid GGDEF proteins were identified: 21 in K. pneumoniae Kp342, 18 in K. pneumoniae MGH 78578 and 17 in K. pneumoniae NTUH-K2044. The majority had only the GGDEF domain, most with the GGEEF motif, and hybrid proteins containing both GGDEF and EAL domains were also found. The I site for allosteric control was identified only in single GGDEF domain proteins and not in hybrid proteins. EAL-only proteins, containing either intact or degenerate domains, were also identified: 15 in Kp342, 15 in MGH 78578 and 10 in NTUH-K2044. Several input sensory domains and transmembrane segments were identified, which together indicate complex regulatory circuits that in many cases can be membrane associated. The comparative analysis of proteins containing GGDEF/EAL domains in K. pneumoniae showed that most copies were shared among the three strains and that some were unique to a particular strain

  2. CelR, an Ortholog of the Diguanylate Cyclase PleD of Caulobacter, Regulates Cellulose Synthesis in Agrobacterium tumefaciens

    PubMed Central

    Barnhart, D. Michael; Su, Shengchang; Baccaro, Brenna E.; Banta, Lois M.

    2013-01-01

    Cellulose fibrils play a role in attachment of Agrobacterium tumefaciens to its plant host. While the genes for cellulose biosynthesis in the bacterium have been identified, little is known concerning the regulation of the process. The signal molecule cyclic di-GMP (c-di-GMP) has been linked to the regulation of exopolysaccharide biosynthesis in many bacterial species, including A. tumefaciens. In this study, we identified two putative diguanylate cyclase genes, celR (atu1297) and atu1060, that influence production of cellulose in A. tumefaciens. Overexpression of either gene resulted in increased cellulose production, while deletion of celR, but not atu1060, resulted in decreased cellulose biosynthesis. celR overexpression also affected other phenotypes, including biofilm formation, formation of a polar adhesion structure, plant surface attachment, and virulence, suggesting that the gene plays a role in regulating these processes. Analysis of celR and Δcel mutants allowed differentiation between phenotypes associated with cellulose production, such as biofilm formation, and phenotypes probably resulting from c-di-GMP signaling, which include polar adhesion, attachment to plant tissue, and virulence. Phylogenetic comparisons suggest that species containing both celR and celA, which encodes the catalytic subunit of cellulose synthase, adapted the CelR protein to regulate cellulose production while those that lack celA use CelR, called PleD, to regulate specific processes associated with polar localization and cell division. PMID:24038703

  3. General Base-General Acid Catalysis by Terpenoid Cyclases§

    PubMed Central

    Pemberton, Travis A.; Christianson, David W.

    2016-01-01

    Terpenoid cyclases catalyze the most complex reactions in biology, in that more than half of the substrate carbon atoms often undergo changes in bonding during the course of a multistep cyclization cascade that proceeds through multiple carbocation intermediates. Many cyclization mechanisms require stereospecific deprotonation and reprotonation steps, and most cyclization cascades are terminated by deprotonation to yield an olefin product. The first bacterial terpenoid cyclase to yield a crystal structure was pentalenene synthase from Streptomyces exfoliatus UC5319. This cyclase generates the hydrocarbon precursor of the pentalenolactone family of antibiotics. The structures of pentalenene synthase and other terpenoid cyclases reveal predominantly nonpolar active sites typically lacking amino acid side chains capable of serving general base-general acid functions. What chemical species, then, enables the Brønsted acid-base chemistry required in the catalytic mechanisms of these enzymes? The most likely candidate for such general base-general acid chemistry is the co-product inorganic pyrophosphate. Here, we briefly review biological and nonbiological systems in which phosphate and its derivatives serve general base and general acid functions in catalysis. These examples highlight the fact that the Brønsted acid-base activities of phosphate derivatives are comparable to the Brønsted acid-base activities of amino acid side chains. PMID:27072285

  4. Cryptic indole hydroxylation by a non-canonical terpenoid cyclase parallels bacterial xenobiotic detoxification

    NASA Astrophysics Data System (ADS)

    Kugel, Susann; Baunach, Martin; Baer, Philipp; Ishida-Ito, Mie; Sundaram, Srividhya; Xu, Zhongli; Groll, Michael; Hertweck, Christian

    2017-06-01

    Terpenoid natural products comprise a wide range of molecular architectures that typically result from C-C bond formations catalysed by classical type I/II terpene cyclases. However, the molecular diversity of biologically active terpenoids is substantially increased by fully unrelated, non-canonical terpenoid cyclases. Their evolutionary origin has remained enigmatic. Here we report the in vitro reconstitution of an unusual flavin-dependent bacterial indoloterpenoid cyclase, XiaF, together with a designated flavoenzyme-reductase (XiaP) that mediates a key step in xiamycin biosynthesis. The crystal structure of XiaF with bound FADH2 (at 2.4 Å resolution) and phylogenetic analyses reveal that XiaF is, surprisingly, most closely related to xenobiotic-degrading enzymes. Biotransformation assays show that XiaF is a designated indole hydroxylase that can be used for the production of indigo and indirubin. We unveil a cryptic hydroxylation step that sets the basis for terpenoid cyclization and suggest that the cyclase has evolved from xenobiotics detoxification enzymes.

  5. A novel zf-MYND protein, CHB-3, mediates guanylyl cyclase localization to sensory cilia and controls body size of Caenorhabditis elegans.

    PubMed

    Fujiwara, Manabi; Teramoto, Takayuki; Ishihara, Takeshi; Ohshima, Yasumi; McIntire, Steven L

    2010-11-24

    Cilia are important sensory organelles, which are thought to be essential regulators of numerous signaling pathways. In Caenorhabditis elegans, defects in sensory cilium formation result in a small-body phenotype, suggesting the role of sensory cilia in body size determination. Previous analyses suggest that lack of normal cilia causes the small-body phenotype through the activation of a signaling pathway which consists of the EGL-4 cGMP-dependent protein kinase and the GCY-12 receptor-type guanylyl cyclase. By genetic suppressor screening of the small-body phenotype of a cilium defective mutant, we identified a chb-3 gene. Genetic analyses placed chb-3 in the same pathway as egl-4 and gcy-12 and upstream of egl-4. chb-3 encodes a novel protein, with a zf-MYND motif and ankyrin repeats, that is highly conserved from worm to human. In chb-3 mutants, GCY-12 guanylyl cyclase visualized by tagged GFP (GCY-12::GFP) fails to localize to sensory cilia properly and accumulates in cell bodies. Our analyses suggest that decreased GCY-12 levels in the cilia of chb-3 mutants may cause the suppression of the small-body phenotype of a cilium defective mutant. By observing the transport of GCY-12::GFP particles along the dendrites to the cilia in sensory neurons, we found that the velocities and the frequencies of the particle movement are decreased in chb-3 mutant animals. How membrane proteins are trafficked to cilia has been the focus of extensive studies in vertebrates and invertebrates, although only a few of the relevant proteins have been identified. Our study defines a new regulator, CHB-3, in the trafficking process and also shows the importance of ciliary targeting of the signaling molecule, GCY-12, in sensory-dependent body size regulation in C. elegans. Given that CHB-3 is highly conserved in mammal, a similar system may be used in the trafficking of signaling proteins to the cilia of other species.

  6. Alpha 2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese hamster ovary cells is mediated by different mechanisms

    SciTech Connect

    Jones, S.B.; Halenda, S.P.; Bylund, D.B.

    1991-02-01

    The effect of alpha 2-adrenergic receptor activation on adenylate cyclase activity in Chinese hamster ovary cells stably transfected with the alpha 2A-adrenergic receptor gene is biphasic. At lower concentrations of epinephrine forskolin-stimulated cyclic AMP production is inhibited, but at higher concentrations the inhibition is reversed. Both of these effects are blocked by the alpha 2 antagonist yohimbine but not by the alpha 1 antagonist prazosin. Pretreatment with pertussis toxin attenuates inhibition at lower concentrations of epinephrine and greatly potentiates forskolin-stimulated cyclic AMP production at higher concentrations of epinephrine. alpha 2-Adrenergic receptor stimulation also causes arachidonic acid mobilization, presumably via phospholipasemore » A2. This effect is blocked by yohimbine, quinacrine, removal of extracellular Ca2+, and pretreatment with pertussis toxin. Quinacrine and removal of extracellular Ca2+, in contrast, have no effect on the enhanced forskolin-stimulated cyclic AMP production. Thus, it appears that the alpha 2-adrenergic receptor in these cells can simultaneously activate distinct signal transduction systems; inhibition of adenylate cyclase and stimulation of phospholipase A2, both via G1, and potentiation of cyclic AMP production by a different (pertussis toxin-insensitive) mechanism.« less

  7. Diguanylate cyclase activity of the Mycobacterium leprae T cell antigen ML1419c

    PubMed Central

    Rotcheewaphan, Suwatchareeporn; Belisle, John T.; Webb, Kristofor J.; Kim, Hee-Jin; Spencer, John S.

    2016-01-01

    The second messenger, bis-(3′,5′)-cyclic dimeric guanosine monophosphate (cyclic di-GMP), is involved in the control of multiple bacterial phenotypes, including those that impact host–pathogen interactions. Bioinformatics analyses predicted that Mycobacterium leprae, an obligate intracellular bacterium and the causative agent of leprosy, encodes three active diguanylate cyclases. In contrast, the related pathogen Mycobacterium tuberculosis encodes only a single diguanylate cyclase. One of the M. leprae unique diguanylate cyclases (ML1419c) was previously shown to be produced early during the course of leprosy. Thus, functional analysis of ML1419c was performed. The gene encoding ML1419c was cloned and expressed in Pseudomonas aeruginosa PAO1 to allow for assessment of cyclic di-GMP production and cyclic di-GMP-mediated phenotypes. Phenotypic studies revealed that ml1419c expression altered colony morphology, motility and biofilm formation of P. aeruginosa PAO1 in a manner consistent with increased cyclic di-GMP production. Direct measurement of cyclic di-GMP levels by liquid chromatography–mass spectrometry confirmed that ml1419c expression increased cyclic di-GMP production in P. aeruginosa PAO1 cultures in comparison to the vector control. The observed phenotypes and increased levels of cyclic di-GMP detected in P. aeruginosa expressing ml1419c could be abrogated by mutation of the active site in ML1419c. These studies demonstrated that ML1419c of M. leprae functions as diguanylate cyclase to synthesize cyclic di-GMP. Thus, this protein was renamed DgcA (Diguanylate cyclase A). These results also demonstrated the ability to use P. aeruginosa as a heterologous host for characterizing the function of proteins involved in the cyclic di-GMP pathway of a pathogen refractory to in vitro growth, M. leprae. PMID:27450520

  8. Diguanylate cyclase activity of the Mycobacterium leprae T cell antigen ML1419c.

    PubMed

    Rotcheewaphan, Suwatchareeporn; Belisle, John T; Webb, Kristofor J; Kim, Hee-Jin; Spencer, John S; Borlee, Bradley R

    2016-09-01

    The second messenger, bis-(3',5')-cyclic dimeric guanosine monophosphate (cyclic di-GMP), is involved in the control of multiple bacterial phenotypes, including those that impact host-pathogen interactions. Bioinformatics analyses predicted that Mycobacterium leprae, an obligate intracellular bacterium and the causative agent of leprosy, encodes three active diguanylate cyclases. In contrast, the related pathogen Mycobacterium tuberculosis encodes only a single diguanylate cyclase. One of the M. leprae unique diguanylate cyclases (ML1419c) was previously shown to be produced early during the course of leprosy. Thus, functional analysis of ML1419c was performed. The gene encoding ML1419c was cloned and expressed in Pseudomonas aeruginosa PAO1 to allow for assessment of cyclic di-GMP production and cyclic di-GMP-mediated phenotypes. Phenotypic studies revealed that ml1419c expression altered colony morphology, motility and biofilm formation of P. aeruginosa PAO1 in a manner consistent with increased cyclic di-GMP production. Direct measurement of cyclic di-GMP levels by liquid chromatography-mass spectrometry confirmed that ml1419c expression increased cyclic di-GMP production in P. aeruginosa PAO1 cultures in comparison to the vector control. The observed phenotypes and increased levels of cyclic di-GMP detected in P. aeruginosa expressing ml1419c could be abrogated by mutation of the active site in ML1419c. These studies demonstrated that ML1419c of M. leprae functions as diguanylate cyclase to synthesize cyclic di-GMP. Thus, this protein was renamed DgcA (Diguanylate cyclase A). These results also demonstrated the ability to use P. aeruginosa as a heterologous host for characterizing the function of proteins involved in the cyclic di-GMP pathway of a pathogen refractory to in vitro growth, M. leprae.

  9. One ring or two? Determination of ring number in carotenoids by lycopene epsilon-cyclases.

    PubMed

    Cunningham, F X; Gantt, E

    2001-02-27

    Carotenoids in the photosynthetic membranes of plants typically contain two beta-rings (e.g., beta-carotene and zeaxanthin) or one epsilon- and one beta-ring (e.g., lutein). Carotenoids with two epsilon-rings are uncommon. We reported earlier that the Arabidopsis thaliana lycopene epsilon-cyclase (LCYe) adds one epsilon-ring to the symmetrical linear substrate lycopene, whereas the structurally related lycopene beta-cyclase (LCYb) adds two beta-rings. Here we describe a cDNA encoding LCYe in romaine lettuce (Lactuca sativa var. romaine), one of the few plant species known to accumulate substantial quantities of a carotenoid with two epsilon-rings: lactucaxanthin. The product of the lettuce cDNA, similar in sequence to the Arabidopsis LCYe (77% amino acid identity), efficiently converted lycopene into the bicyclic epsilon-carotene in a heterologous Escherichia coli system. Regions of the lettuce and Arabidopsis epsilon-cyclases involved in the determination of ring number were mapped by analysis of chimeric epsilon-cyclases constructed by using an inverse PCR approach. A single amino acid was found to act as a molecular switch: lettuce LCYe mutant H457L added only one epsilon-ring to lycopene, whereas the complementary Arabidopsis LCYe mutant, L448H, added two epsilon-rings. An R residue in this position also yields a bi-epsilon-cyclase for both the lettuce and Arabidopsis enzymes. Construction and analysis of chimera of related enzymes with differing catalytic activities provide an informative approach that may be of particular utility for studying membrane-associated enzymes that cannot easily be crystallized or modeled to existing crystal structures.

  10. One ring or two? Determination of ring number in carotenoids by lycopene ɛ-cyclases

    PubMed Central

    Cunningham, Francis X.; Gantt, Elisabeth

    2001-01-01

    Carotenoids in the photosynthetic membranes of plants typically contain two β-rings (e.g., β-carotene and zeaxanthin) or one ɛ- and one β-ring (e.g., lutein). Carotenoids with two ɛ-rings are uncommon. We reported earlier that the Arabidopsis thaliana lycopene ɛ-cyclase (LCYe) adds one ɛ-ring to the symmetrical linear substrate lycopene, whereas the structurally related lycopene β-cyclase (LCYb) adds two β-rings. Here we describe a cDNA encoding LCYe in romaine lettuce (Lactuca sativa var. romaine), one of the few plant species known to accumulate substantial quantities of a carotenoid with two ɛ-rings: lactucaxanthin. The product of the lettuce cDNA, similar in sequence to the Arabidopsis LCYe (77% amino acid identity), efficiently converted lycopene into the bicyclic ɛ-carotene in a heterologous Escherichia coli system. Regions of the lettuce and Arabidopsis ɛ-cyclases involved in the determination of ring number were mapped by analysis of chimeric ɛ-cyclases constructed by using an inverse PCR approach. A single amino acid was found to act as a molecular switch: lettuce LCYe mutant H457L added only one ɛ-ring to lycopene, whereas the complementary Arabidopsis LCYe mutant, L448H, added two ɛ-rings. An R residue in this position also yields a bi-ɛ-cyclase for both the lettuce and Arabidopsis enzymes. Construction and analysis of chimera of related enzymes with differing catalytic activities provide an informative approach that may be of particular utility for studying membrane-associated enzymes that cannot easily be crystallized or modeled to existing crystal structures. PMID:11226339

  11. 5-HT stimulation of heart rate in Drosophila does not act through cAMP as revealed by pharmacogenetics.

    PubMed

    Majeed, Zana R; Nichols, Charles D; Cooper, Robin L

    2013-12-01

    The fruit fly, Drosophila melanogaster, is a good experimental organism to study the underlying mechanism of heart rate (HR) regulation. It is already known that many neuromodulators (serotonin, dopamine, octopamine, acetylcholine) change the HR in Drosophila melanogaster larvae. In this study, we investigated the role of cAMP-PKA signaling pathway in HR regulation and 5-HT positive chronotropic action. In order to obtain insight into the 5-HT mechanism of action in larvae cardiomyocytes, genetic and pharmacological approaches were used. We used transgenic flies that expressed the hM4Di receptor [designer receptors exclusively activated by designer drugs (DREADDs)] as one tool. Our previous results showed that activation of hM4Di receptors (modified muscarinic acetylcholine receptors) decreases or arrests the heart from beating. In this study, it was hypothesized that the positive chronotropic effect of serotonin [5-hydroxytryptamine (5-HT)] are mediated by serotonin receptors coupled to the adenylyl cyclase pathway and downstream cAMP and PKA activity. Activation of hM4Di by clozapine-N-oxide (CNO) was predicted to block the effects of serotonin by inhibiting adenylyl cyclase activity through Gαi pathway activation. Interestingly, we found here that manipulation of adenylyl cyclase activity and cAMP levels had no significant effect on HR. The ability of hM4Di receptor activation to slow or stop the heart is therefore likely mediated by activation of GIRK channels to produce hyperpolarization of cardiomyocytes, and not through inhibition of adenylyl cyclase.

  12. Kinetic Analysis of DNA Strand Joining by Chlorella Virus DNA Ligase and the Role of Nucleotidyltransferase Motif VI in Ligase Adenylylation*

    PubMed Central

    Samai, Poulami; Shuman, Stewart

    2012-01-01

    Chlorella virus DNA ligase (ChVLig) is an instructive model for mechanistic studies of the ATP-dependent DNA ligase family. ChVLig seals 3′-OH and 5′-PO4 termini via three chemical steps: 1) ligase attacks the ATP α phosphorus to release PPi and form a covalent ligase-adenylate intermediate; 2) AMP is transferred to the nick 5′-phosphate to form DNA-adenylate; 3) the 3′-OH of the nick attacks DNA-adenylate to join the polynucleotides and release AMP. Each chemical step requires Mg2+. Kinetic analysis of nick sealing by ChVLig-AMP revealed that the rate constant for phosphodiester synthesis (kstep3 = 25 s−1) exceeds that for DNA adenylylation (kstep2 = 2.4 s−1) and that Mg2+ binds with similar affinity during step 2 (Kd = 0.77 mm) and step 3 (Kd = 0.87 mm). The rates of DNA adenylylation and phosphodiester synthesis respond differently to pH, such that step 3 becomes rate-limiting at pH ≤ 6.5. The pH profiles suggest involvement of one and two protonation-sensitive functional groups in catalysis of steps 2 and 3, respectively. We suggest that the 5′-phosphate of the nick is the relevant protonation-sensitive moiety and that a dianionic 5′-phosphate is necessary for productive step 2 catalysis. Motif VI, located at the C terminus of the OB-fold domain of ChVLig, is a conserved feature of ATP-dependent DNA ligases and GTP-dependent mRNA capping enzymes. Presteady state and burst kinetic analysis of the effects of deletion and missense mutations highlight the catalytic contributions of ChVLig motif VI, especially the Asp-297 carboxylate, exclusively during the ligase adenylylation step. PMID:22745124

  13. Presynaptic Dopamine D2 Receptors Modulate [3H]GABA Release at StriatoPallidal Terminals via Activation of PLC → IP3 → Calcineurin and Inhibition of AC → cAMP → PKA Signaling Cascades.

    PubMed

    Jijón-Lorenzo, Rafael; Caballero-Florán, Isaac Hiram; Recillas-Morales, Sergio; Cortés, Hernán; Avalos-Fuentes, José Arturo; Paz-Bermúdez, Francisco Javier; Erlij, David; Florán, Benjamín

    2018-02-21

    Striatal dopamine D2 receptors activate the PLC → IP3 → Calcineurin-signaling pathway to modulate the neural excitability of En+ Medium-sized Spiny GABAergic neurons (MSN) through the regulation of L-type Ca 2+ channels. Presynaptic dopaminergic D2 receptors modulate GABA release at striatopallidal terminals through L-type Ca 2+ channels as well, but their signaling pathway is still undetermined. Since D2 receptors are Gi/o-coupled and negatively modulate adenylyl cyclase (AC), we investigated whether presynaptic D2 receptors modulate GABA release through the same signaling cascade that controls excitability in the striatum or by the inhibition of AC and decreased PKA activity. Activation of D2 receptors stimulated formation of [ 3 H]IP 1 and decreased Forskolin-stimulated [ 3 H]cAMP accumulation in synaptosomes from rat Globus Pallidus. D2 receptor activation with Quinpirole in the presence of L 745,870 decreased, in a dose-dependent manner, K + -induced [ 3 H]GABA release in pallidal slices. The effect was prevented by the pharmacological blockade of Gi/o βγ subunit effects with Gallein, PLC with U 73122, IP3 receptor activation with 4-APB, Calcineurin with FK506. In addition, when release was stimulated with Forskolin to activate AC, D2 receptors also decreased K + -induced [ 3 H]GABA release, an effect occluded with the effect of the blockade of PKA with H89 or stimulation of release with the cAMP analog 8-Br-cAMP. These data indicate that D2 receptors modulate [ 3 H]GABA release at striatopallidal terminals by activating the PLC → IP3 → Calcineurin-signaling cascade, the same one that modulates excitability in soma. Additionally, D2 receptors inhibit release when AC is active. Both mechanisms appear to converge to regulate the activity of presynaptic L-type Ca 2+ channels. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

  14. Biotin increases glucokinase expression via soluble guanylate cyclase/protein kinase G, adenosine triphosphate production and autocrine action of insulin in pancreatic rat islets.

    PubMed

    Vilches-Flores, Alonso; Tovar, Armando R; Marin-Hernandez, Alvaro; Rojas-Ochoa, Alberto; Fernandez-Mejia, Cristina

    2010-07-01

    Besides its role as a carboxylase prosthetic group, biotin has important effects on gene expression. However, the molecular mechanisms through which biotin exerts these effects are largely unknown. We previously found that biotin increases pancreatic glucokinase expression. We have now explored the mechanisms underlying this effect. Pancreatic islets from Wistar rats were treated with biotin, in the presence or absence of different types of inhibitors. Glucokinase mRNA and 18s rRNA abundance were determined by real-time PCR. Adenosine triphosphate (ATP) content was analyzed by fluorometry. Biotin treatment increased glucokinase mRNA abundance approximately one fold after 2 h; the effect was sustained up to 24 h. Inhibition of soluble guanylate cyclase or protein kinase G (PKG) signalling suppressed biotin-induced glucokinase expression. The cascade of events downstream of PKG in biotin-mediated gene transcription is not known. We found that inhibition of insulin secretion with diazoxide or nifedipine prevented biotin-stimulated glucokinase mRNA increase. Biotin treatment increased islet ATP content (control: 4.68+/-0.28; biotin treated: 6.62+/-0.26 pmol/islet) at 30 min. Inhibition of PKG activity suppressed the effects of biotin on ATP content. Insulin antibodies or inhibitors of phosphoinositol-3-kinase/Akt insulin signalling pathway prevented biotin-induced glucokinase expression. The nucleotide 8-Br-cGMP mimicked the biotin effects. We propose that the induction of pancreatic glucokinase mRNA by biotin involves guanylate cyclase and PKG activation, which leads to an increase in ATP content. This induces insulin secretion via ATP-sensitive potassium channels. Autocrine insulin, in turn, activates phosphoinositol-3-kinase/Akt signalling. Our results offer new insights into the pathways that participate in biotin-mediated gene expression. (c) 2010 Elsevier Inc. All rights reserved.

  15. Expression of membrane-bound and cytosolic guanylyl cyclases in the rat inner ear.

    PubMed

    Seebacher, T; Beitz, E; Kumagami, H; Wild, K; Ruppersberg, J P; Schultz, J E

    1999-01-01

    Membrane-bound guanylyl cyclases (GCs) are peptide hormone receptors whereas the cytosolic isoforms are receptors for nitric oxide. In the inner ear, the membrane-bound GCs may be involved in the regulation of fluid homeostasis and the cytosolic forms possibly play a role in signal processing and regulation of local blood flow. In this comprehensive study, we examined, qualitatively and quantitatively, the transcription pattern of all known GC isoforms in the inner ear from rat by RT-PCR. The tissues used were endolymphatic sac, stria vascularis, organ of Corti, organ of Corti outer hair cells, cochlear nerve, Reissner's membrane, vestibular dark cells, and vestibular sensory cells. We show that multiple particulate (GC-A, GC-B, GC-D, GC-E, GC-F and GC-G) and several subunits of the heterodimeric cytosolic GCs (alpha1, alpha2, beta1 and beta2) are expressed, albeit at highly different levels. GC-C was not found. GC-A and the soluble subunits alpha1 and beta1 were transcribed ubiquitously. GC-B was present in all tissues except stria vascularis, which contained GC-A and traces of GC-E and GC-G. GC-B was by far the predominant membrane-bound isoform in the organ of Corti (86%), Reissner's membrane (75%) and the vestibulum (80%). Surprisingly, GC-E, a retinal isoform, was detected in significant amounts in the cochlear nerve (8%) and in the organ of Corti (4%). Although the cytosolic GC is a heterodimer composed of an alpha and a beta subunit, the mRNA transcription of these subunits was not stoichiometric. Particularly in the vestibulum, the transcription of the beta1 subunits was at least four-fold higher than of the alpha1 subunit. The data are compatible with earlier suggestions that membrane receptor GCs may be involved in the control of inner ear electrolyte and fluid composition whereas NO-stimulated GC isoforms mainly participate in the regulation of blood flow and supporting cell physiology.

  16. The Arrestin-selective Angiotensin AT1 Receptor Agonist [Sar1,Ile4,Ile8]-AngII Negatively Regulates Bradykinin B2 Receptor Signaling via AT1-B2 Receptor Heterodimers*

    PubMed Central

    Wilson, Parker C.; Lee, Mi-Hye; Appleton, Kathryn M.; El-Shewy, Hesham M.; Morinelli, Thomas A.; Peterson, Yuri K.; Luttrell, Louis M.; Jaffa, Ayad A.

    2013-01-01

    The renin-angiotensin and kallikrein-kinin systems are key regulators of vascular tone and inflammation. Angiotensin II, the principal effector of the renin-angiotensin system, promotes vasoconstriction by activating angiotensin AT1 receptors. The opposing effects of the kallikrein-kinin system are mediated by bradykinin acting on B1 and B2 bradykinin receptors. The renin-angiotensin and kallikrein-kinin systems engage in cross-talk at multiple levels, including the formation of AT1-B2 receptor heterodimers. In primary vascular smooth muscle cells, we find that the arrestin pathway-selective AT1 agonist, [Sar1,Ile4,Ile8]-AngII, but not the neutral AT1 antagonist, losartan, inhibits endogenous B2 receptor signaling. In a transfected HEK293 cell model that recapitulates this effect, we find that the actions of [Sar1,Ile4, Ile8]-AngII require the AT1 receptor and result from arrestin-dependent co-internalization of AT1-B2 heterodimers. BRET50 measurements indicate that AT1 and B2 receptors efficiently heterodimerize. In cells expressing both receptors, pretreatment with [Sar1,Ile4,Ile8]-AngII blunts B2 receptor activation of Gq/11-dependent intracellular calcium influx and Gi/o-dependent inhibition of adenylyl cyclase. In contrast, [Sar1,Ile4,Ile8]-AngII has no effect on B2 receptor ligand affinity or bradykinin-induced arrestin3 recruitment. Both radioligand binding assays and quantitative microscopy-based analysis demonstrate that [Sar1,Ile4,Ile8]-AngII promotes internalization of AT1-B2 heterodimers. Thus, [Sar1,Ile4,Ile8]-AngII exerts lateral allosteric modulation of B2 receptor signaling by binding to the orthosteric ligand binding site of the AT1 receptor and promoting co-sequestration of AT1-B2 heterodimers. Given the opposing roles of the renin-angiotensin and kallikrein-kinin systems in vivo, the distinct properties of arrestin pathway-selective and neutral AT1 receptor ligands may translate into different pharmacologic actions. PMID:23661707

  17. The arrestin-selective angiotensin AT1 receptor agonist [Sar1,Ile4,Ile8]-AngII negatively regulates bradykinin B2 receptor signaling via AT1-B2 receptor heterodimers.

    PubMed

    Wilson, Parker C; Lee, Mi-Hye; Appleton, Kathryn M; El-Shewy, Hesham M; Morinelli, Thomas A; Peterson, Yuri K; Luttrell, Louis M; Jaffa, Ayad A

    2013-06-28

    The renin-angiotensin and kallikrein-kinin systems are key regulators of vascular tone and inflammation. Angiotensin II, the principal effector of the renin-angiotensin system, promotes vasoconstriction by activating angiotensin AT1 receptors. The opposing effects of the kallikrein-kinin system are mediated by bradykinin acting on B1 and B2 bradykinin receptors. The renin-angiotensin and kallikrein-kinin systems engage in cross-talk at multiple levels, including the formation of AT1-B2 receptor heterodimers. In primary vascular smooth muscle cells, we find that the arrestin pathway-selective AT1 agonist, [Sar(1),Ile(4),Ile(8)]-AngII, but not the neutral AT1 antagonist, losartan, inhibits endogenous B2 receptor signaling. In a transfected HEK293 cell model that recapitulates this effect, we find that the actions of [Sar(1),Ile(4), Ile(8)]-AngII require the AT1 receptor and result from arrestin-dependent co-internalization of AT1-B2 heterodimers. BRET50 measurements indicate that AT1 and B2 receptors efficiently heterodimerize. In cells expressing both receptors, pretreatment with [Sar(1),Ile(4),Ile(8)]-AngII blunts B2 receptor activation of Gq/11-dependent intracellular calcium influx and Gi/o-dependent inhibition of adenylyl cyclase. In contrast, [Sar(1),Ile(4),Ile(8)]-AngII has no effect on B2 receptor ligand affinity or bradykinin-induced arrestin3 recruitment. Both radioligand binding assays and quantitative microscopy-based analysis demonstrate that [Sar(1),Ile(4),Ile(8)]-AngII promotes internalization of AT1-B2 heterodimers. Thus, [Sar(1),Ile(4),Ile(8)]-AngII exerts lateral allosteric modulation of B2 receptor signaling by binding to the orthosteric ligand binding site of the AT1 receptor and promoting co-sequestration of AT1-B2 heterodimers. Given the opposing roles of the renin-angiotensin and kallikrein-kinin systems in vivo, the distinct properties of arrestin pathway-selective and neutral AT1 receptor ligands may translate into different pharmacologic

  18. Dysfunctional nitric oxide signalling increases risk of myocardial infarction.

    PubMed

    Erdmann, Jeanette; Stark, Klaus; Esslinger, Ulrike B; Rumpf, Philipp Moritz; Koesling, Doris; de Wit, Cor; Kaiser, Frank J; Braunholz, Diana; Medack, Anja; Fischer, Marcus; Zimmermann, Martina E; Tennstedt, Stephanie; Graf, Elisabeth; Eck, Sebastian; Aherrahrou, Zouhair; Nahrstaedt, Janja; Willenborg, Christina; Bruse, Petra; Brænne, Ingrid; Nöthen, Markus M; Hofmann, Per; Braund, Peter S; Mergia, Evanthia; Reinhard, Wibke; Burgdorf, Christof; Schreiber, Stefan; Balmforth, Anthony J; Hall, Alistair S; Bertram, Lars; Steinhagen-Thiessen, Elisabeth; Li, Shu-Chen; März, Winfried; Reilly, Muredach; Kathiresan, Sekar; McPherson, Ruth; Walter, Ulrich; Ott, Jurg; Samani, Nilesh J; Strom, Tim M; Meitinger, Thomas; Hengstenberg, Christian; Schunkert, Heribert

    2013-12-19

    Myocardial infarction, a leading cause of death in the Western world, usually occurs when the fibrous cap overlying an atherosclerotic plaque in a coronary artery ruptures. The resulting exposure of blood to the atherosclerotic material then triggers thrombus formation, which occludes the artery. The importance of genetic predisposition to coronary artery disease and myocardial infarction is best documented by the predictive value of a positive family history. Next-generation sequencing in families with several affected individuals has revolutionized mutation identification. Here we report the segregation of two private, heterozygous mutations in two functionally related genes, GUCY1A3 (p.Leu163Phefs*24) and CCT7 (p.Ser525Leu), in an extended myocardial infarction family. GUCY1A3 encodes the α1 subunit of soluble guanylyl cyclase (α1-sGC), and CCT7 encodes CCTη, a member of the tailless complex polypeptide 1 ring complex, which, among other functions, stabilizes soluble guanylyl cyclase. After stimulation with nitric oxide, soluble guanylyl cyclase generates cGMP, which induces vasodilation and inhibits platelet activation. We demonstrate in vitro that mutations in both GUCY1A3 and CCT7 severely reduce α1-sGC as well as β1-sGC protein content, and impair soluble guanylyl cyclase activity. Moreover, platelets from digenic mutation carriers contained less soluble guanylyl cyclase protein and consequently displayed reduced nitric-oxide-induced cGMP formation. Mice deficient in α1-sGC protein displayed accelerated thrombus formation in the microcirculation after local trauma. Starting with a severely affected family, we have identified a link between impaired soluble-guanylyl-cyclase-dependent nitric oxide signalling and myocardial infarction risk, possibly through accelerated thrombus formation. Reversing this defect may provide a new therapeutic target for reducing the risk of myocardial infarction.

  19. Reduced sensitivity of the hepatic adenylate cyclase-cyclic AMP system to glucagon during sustained hormonal stimulation.

    PubMed Central

    DeRubertis, F R; Craven, P

    1976-01-01

    Hormone-induced desensitization of hormonal regulation of cyclic AMP (cAMP) content has been described in a number of tissues. In the present study, we examined responses of rat liver to glucagon after periods of sustained exposure to the hormone in vivo and in vitro. In intact anesthetized rats infused with glucagon (50 ng/min) for 1 h or more and in liver slices incubated with the hormone (10 muM) for this period, hepatic cAMP responsiveness to glucagon was significantly blunted compared with that of tissue exposed to the hormone for shorter periods. The reduction in hepatic cAMP responsiveness to glucagon appeared to be fully expressed by 2 h. With the doses of hormone employed, the sequential alterations in hepatic responsiveness seemed to be limited to the cAMP system, since other parameters of glucagon action did not wane with time. Diminished hepatic cAMP responsiveness during sustained hormonal exposure could not be attributed to decreased glucagon availability, accelerated extracellular release of cAMP, hepatic ATP depletion, or enhanced phosphodiesterase activity. Studies in vitro suggested that modulation of the cAMP response occurred at the level of adenylate cyclase (AC). During sustained exposure of hepatic slices to glucagon, reductions in glucagon-responsive AC correlated temporally with those in cAMP and both changes were reversible. Alterations in glucagon-responsive AC were demonstrated over a wide range of ATP (10 muM-0.1 mM) and glucagon (10 nM-5 MM) concentrations in the cyclase reaction mixture, and appeared to be a noncompetitive phenomenon relative to glucagon. Maximal NaF-responsive AC did not fall concomitantly with time. Thus, the reduction in glucagon-responsive AC was probably not related to a reduction in the catalytic unit of the enzyme, but could have been due to an alteration in glucagon binding to its receptor sites, or in the coupling mechanism involved in transmission of the hormonal signal to the catalytic unit. Images PMID

  20. Aspartate 102 in the Heme Domain of Soluble Guanylyl Cyclase Has a Key Role in NO Activation

    PubMed Central

    Baskaran, Padmamalini; Heckler, Erin J.; van den Akker, Focco; Beuve, Annie

    2012-01-01

    Nitric oxide (NO) is involved in the physiology and pathophysiology of the cardiovascular and neuronal systems via activation of soluble guanylyl cyclase (sGC), a heme-containing heterodimer. Recent structural studies have allowed a better understanding of the residues that dictate the affinity and binding of NO to the heme and the resulting breakage of the bond between the heme iron and histidine 105 (H105) of the β subunit of sGC. Still, it is unknown how the breakage of the iron–His bond translates into NO-dependent increased catalysis. Structural studies on homologous H-NOX domains in various states pointed to a role for movement of the H105 containing αF helix. Our modeling of the heme-binding domain highlighted conserved residues in the vicinity of H105 that could potentially regulate the extent to which the αF helix shifts and/or propagate the activation signal once the covalent bond with H105 has been broken. These include a direct interaction of αF helix residue D102 with the backbone nitrogen of F120. Mutational analysis of this region points to an essential role of the interactions in the vicinity of H105 for heme stability and identifies aspartate 102 (D102) as having a key role in NO activation following breakage of the iron–His bond. PMID:21491881

  1. Snf1 Phosphorylates Adenylate Cyclase and Negatively Regulates Protein Kinase A-dependent Transcription in Saccharomyces cerevisiae.

    PubMed

    Nicastro, Raffaele; Tripodi, Farida; Gaggini, Marco; Castoldi, Andrea; Reghellin, Veronica; Nonnis, Simona; Tedeschi, Gabriella; Coccetti, Paola

    2015-10-09

    In eukaryotes, nutrient availability and metabolism are coordinated by sensing mechanisms and signaling pathways, which influence a broad set of cellular functions such as transcription and metabolic pathways to match environmental conditions. In yeast, PKA is activated in the presence of high glucose concentrations, favoring fast nutrient utilization, shutting down stress responses, and boosting growth. On the contrary, Snf1/AMPK is activated in the presence of low glucose or alternative carbon sources, thus promoting an energy saving program through transcriptional activation and phosphorylation of metabolic enzymes. The PKA and Snf1/AMPK pathways share common downstream targets. Moreover, PKA has been reported to negatively influence the activation of Snf1/AMPK. We report a new cross-talk mechanism with a Snf1-dependent regulation of the PKA pathway. We show that Snf1 and adenylate cyclase (Cyr1) interact in a nutrient-independent manner. Moreover, we identify Cyr1 as a Snf1 substrate and show that Snf1 activation state influences Cyr1 phosphorylation pattern, cAMP intracellular levels, and PKA-dependent transcription. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Pituitary adenylate cyclase-activating polypeptide type 1 (PAC1) receptor is expressed during embryonic development of the earthworm.

    PubMed

    Boros, Akos; Somogyi, Ildikó; Engelmann, Péter; Lubics, Andrea; Reglodi, Dóra; Pollák, Edit; Molnár, László

    2010-03-01

    Pituitary adenylate cyclase activating polypeptide (PACAP)-like molecules have been shown to be present in cocoon albumin and in Eisenia fetida embryos at an early developmental stage (E1) by immunocytochemistry and radioimmunoassay. Here, we focus on detecting the stage at which PAC1 receptor (PAC1R)-like immunoreactivity first appears in germinal layers and structures, e.g., various parts of the central nervous system (CNS), in developing earthworm embryos. PAC1R-like immunoreactivity was revealed by Western blot and Far Western blot as early as the E2 developmental stage, occurring in the ectoderm and later in specific neurons of the developing CNS. Labeled CNS neurons were first seen in the supraesophageal ganglion (brain) and subsequently in the subesophageal and ventral nerve cord ganglia. Ultrastructurally, PAC1Rs were located mainly on plasma membranes and intracellular membranes, especially on cisternae of the endoplasmic reticulum. Therefore, PACAP-like compounds probably influence the differentiation of germinal layers (at least the ectoderm) and of some neurons and might act as signaling molecules during earthworm embryonic development.

  3. c-di-GMP Turn-Over in Clostridium difficile Is Controlled by a Plethora of Diguanylate Cyclases and Phosphodiesterases

    PubMed Central

    Bordeleau, Eric; Fortier, Louis-Charles; Malouin, François; Burrus, Vincent

    2011-01-01

    Clostridium difficile infections have become a major healthcare concern in the last decade during which the emergence of new strains has underscored this bacterium's capacity to cause persistent epidemics. c-di-GMP is a bacterial second messenger regulating diverse bacterial phenotypes, notably motility and biofilm formation, in proteobacteria such as Vibrio cholerae, Pseudomonas aeruginosa, and Salmonella. c-di-GMP is synthesized by diguanylate cyclases (DGCs) that contain a conserved GGDEF domain. It is degraded by phosphodiesterases (PDEs) that contain either an EAL or an HD-GYP conserved domain. Very little is known about the role of c-di-GMP in the regulation of phenotypes of Gram-positive or fastidious bacteria. Herein, we exposed the main components of c-di-GMP signalling in 20 genomes of C. difficile, revealed their prevalence, and predicted their enzymatic activity. Ectopic expression of 31 of these conserved genes was carried out in V. cholerae to evaluate their effect on motility and biofilm formation, two well-characterized phenotype alterations associated with intracellular c-di-GMP variation in this bacterium. Most of the predicted DGCs and PDEs were found to be active in the V. cholerae model. Expression of truncated versions of CD0522, a protein with two GGDEF domains and one EAL domain, suggests that it can act alternatively as a DGC or a PDE. The activity of one purified DGC (CD1420) and one purified PDE (CD0757) was confirmed by in vitro enzymatic assays. GTP was shown to be important for the PDE activity of CD0757. Our results indicate that, in contrast to most Gram-positive bacteria including its closest relatives, C. difficile encodes a large assortment of functional DGCs and PDEs, revealing that c-di-GMP signalling is an important and well-conserved signal transduction system in this human pathogen. PMID:21483756

  4. Convergent phosphomodulation of the major neuronal dendritic potassium channel Kv4.2 by pituitary adenylate cyclase-activating polypeptide.

    PubMed

    Gupte, Raeesa P; Kadunganattil, Suraj; Shepherd, Andrew J; Merrill, Ronald; Planer, William; Bruchas, Michael R; Strack, Stefan; Mohapatra, Durga P

    2016-02-01

    The endogenous neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is secreted by both neuronal and non-neuronal cells in the brain and spinal cord, in response to pathological conditions such as stroke, seizures, chronic inflammatory and neuropathic pain. PACAP has been shown to exert various neuromodulatory and neuroprotective effects. However, direct influence of PACAP on the function of intrinsically excitable ion channels that are critical to both hyperexcitation as well as cell death, remain largely unexplored. The major dendritic K(+) channel Kv4.2 is a critical regulator of neuronal excitability, back-propagating action potentials in the dendrites, and modulation of synaptic inputs. We identified, cloned and characterized the downstream signaling originating from the activation of three PACAP receptor (PAC1) isoforms that are expressed in rodent hippocampal neurons that also exhibit abundant expression of Kv4.2 protein. Activation of PAC1 by PACAP leads to phosphorylation of Kv4.2 and downregulation of channel currents, which can be attenuated by inhibition of either PKA or ERK1/2 activity. Mechanistically, this dynamic downregulation of Kv4.2 function is a consequence of reduction in the density of surface channels, without any influence on the voltage-dependence of channel activation. Interestingly, PKA-induced effects on Kv4.2 were mediated by ERK1/2 phosphorylation of the channel at two critical residues, but not by direct channel phosphorylation by PKA, suggesting a convergent phosphomodulatory signaling cascade. Altogether, our findings suggest a novel GPCR-channel signaling crosstalk between PACAP/PAC1 and Kv4.2 channel in a manner that could lead to neuronal hyperexcitability. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase.

    PubMed

    Bauer, Robert J; Evans, Thomas C; Lohman, Gregory J S

    2016-01-01

    DNA ligases are essential both to in vivo replication, repair and recombination processes, and in vitro molecular biology protocols. Prior characterization of DNA ligases through gel shift assays has shown the presence of a nick site to be essential for tight binding between the enzyme and its dsDNA substrate, with no interaction evident on dsDNA lacking a nick. In the current study, we observed a significant substrate inhibition effect, as well as the inhibition of both the self-adenylylation and nick-sealing steps of T4 DNA ligase by non-nicked, non-substrate dsDNA. Inhibition by non-substrate DNA was dependent only on the total DNA concentration rather than the structure; with 1 μg/mL of 40-mers, 75-mers, or circular plasmid DNA all inhibiting ligation equally. A >15-fold reduction in T4 DNA ligase self-adenylylation rate when in the presence of high non-nicked dsDNA concentrations was observed. Finally, EMSAs were utilized to demonstrate that non-substrate dsDNA can compete with nicked dsDNA substrates for enzyme binding. Based upon these data, we hypothesize the inhibition of T4 DNA ligase by non-nicked dsDNA is direct evidence for a two-step nick-binding mechanism, with an initial, nick-independent, transient dsDNA-binding event preceding a transition to a stable binding complex in the presence of a nick site.

  6. The Inhibitory Effect of Non-Substrate and Substrate DNA on the Ligation and Self-Adenylylation Reactions Catalyzed by T4 DNA Ligase

    PubMed Central

    Bauer, Robert J.; Evans, Thomas C.; Lohman, Gregory J. S.

    2016-01-01

    DNA ligases are essential both to in vivo replication, repair and recombination processes, and in vitro molecular biology protocols. Prior characterization of DNA ligases through gel shift assays has shown the presence of a nick site to be essential for tight binding between the enzyme and its dsDNA substrate, with no interaction evident on dsDNA lacking a nick. In the current study, we observed a significant substrate inhibition effect, as well as the inhibition of both the self-adenylylation and nick-sealing steps of T4 DNA ligase by non-nicked, non-substrate dsDNA. Inhibition by non-substrate DNA was dependent only on the total DNA concentration rather than the structure; with 1 μg/mL of 40-mers, 75-mers, or circular plasmid DNA all inhibiting ligation equally. A >15-fold reduction in T4 DNA ligase self-adenylylation rate when in the presence of high non-nicked dsDNA concentrations was observed. Finally, EMSAs were utilized to demonstrate that non-substrate dsDNA can compete with nicked dsDNA substrates for enzyme binding. Based upon these data, we hypothesize the inhibition of T4 DNA ligase by non-nicked dsDNA is direct evidence for a two-step nick-binding mechanism, with an initial, nick-independent, transient dsDNA-binding event preceding a transition to a stable binding complex in the presence of a nick site. PMID:26954034

  7. Localization of soluble guanylate cyclase activity in the guinea pig inner ear.

    PubMed

    Takumida, M; Anniko, M; Popa, R; Zhang, D M

    2000-01-01

    The aim of this study was to characterize the nitric oxide (NO) receptor soluble guanylate cyclase (sGC), to determine the cells targeted by NO and to elucidate the function of the NO/cGMP pathway in the inner ear. sGC activity in the inner ear was localized by immunohistochemical detection of NO-stimulated cGMP. Soluble guanylate cyclase activity in the cochlea was detected in the nerve endings underneath the outer and inner hair cells, supporting cells, stria vascularis and vessels. In the vestibular organs, sGC activity was detected in the cytoplasm of sensory cells, nerve fibres, dark cells and transitional cells and vessels. These findings suggest that the NO/cGMP pathway may be involved in regulatory processes in neurotransmission, blood flow and inner ear fluid homeostasis.

  8. In vivo adenylate cyclase activity in ultraviolet- and gamma-irradiated Escherichia coli.

    PubMed

    Chatterjee, A; Bhattacharya, A K

    1988-06-01

    The incorporation of [14C]adenine into the cyclic AMP fraction by whole cells of Escherichia coli B/r was taken as a measure of the in vivo adenylate cyclase activity. This activity was significantly inhibited by irradiation of the cells either with 60Co gamma-rays or with UV light from a germicidal lamp, suggesting inhibition of cyclic AMP synthesis. The incubation of cells after irradiation with lower doses (50-100 Gy) of gamma-rays produced a significant increase of in vivo adenylate cyclase activity, whereas there was no significant change after higher doses (150 Gy and above). Dark incubation of cells after irradiation with UV light (54 J m-2) led to recovery of enzyme activity to the level measured in unirradiated cells. Thus it appears that the catabolite repression of L-arabinose isomerase induced by UV light, as well as gamma-irradiation, is due to reduced cyclic AMP synthesis in irradiated cells.

  9. Influence of volatile anesthetics on muscarinic receptor adenylate cyclase coupling in brain and heart

    SciTech Connect

    Anthony, B.L.

    In the present study, the influence of four volatile anesthetics (enflurane, isoflurane, diethyl ether, and chloroform) on (1) muscarinic receptor binding parameters and (2) muscarnic regulation of adenylate cyclase activity was examined using membranes isolated from rat brain and heart. Membranes were equilibrated with each of the four anesthetics for 30 minutes and then during the binding assay. The data obtained can be summarized as follows: (1) volatile anesthetics increased receptor affinity for a radiolabeled antagonists, ({sup 3}H)N-methylscopolamine (({sup 3}H)MS), by decreasing its rate of dissociation in brain stem, but not in cardiac, membranes, (2) volatile anesthetics decreased high affinitymore » ({sup 3}H)Oxotremorine-M binding, (3) volatile anesthetics depressed or eliminated the guanine nucleotide sensitivity of agonist binding. The influence of volatile anesthetics on muscarinic regulation of adenylate cyclase enzyme activity was studied using {alpha}({sup 32}P)ATP as the substrate.« less

  10. Antagonism of histamine-activated adenylate cyclase in brain by D-lysergic acid diethylamide.

    PubMed

    Green, J P; Johnson, C L; Weinstein, H; Maayani, S

    1977-12-01

    D-Lysergic acid diethylamide and D-2-bromolysergic acid diethylamide are competitive antagonists of the histamine activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); E.C. 4.6.1.1] in broken cell preparations of the hippocampus and cortex of guinea pig brain. The adenylate cyclase is linked to the histamine H2-receptor. Both D-lysergic acid diethylamide and D-2-bromolysergic acid diethylamide show topological congruency with potent H2-antagonists. D-2-Bromolysergic acid diethylamide is 10 times more potent as an H2-antagonist than cimetidine, which has been the most potent H2-antagonist reported, and D-lysergic acid diethylamide is about equipotent to cimetidine. Blockade of H2-receptors could contribute to the behavioral effects of D-2-bromolysergic acid diethylamide and D-lysergic acid diethylamide.

  11. Effect of hypolipidemic drugs on basal and stimulated adenylate cyclase activity in tumor cells

    SciTech Connect

    Bershtein, L.M.; Kovaleva, I.G.; Rozenberg, O.A.

    1986-02-01

    This paper studies adenylate cyclase acticvity in Ehrlich's ascites carcinoma (EAC) cells during administration of drugs with a hypolipidemic action. Seven to eight days before they were killed, male mice ingested the antidiabetic biguanide phenformin, and the phospholipid-containing preparation Essentiale in drinking water. The cAMP formed was isolated by chromatography on Silufol plates after incubation of the enzyme preparation with tritium-ATP, or was determined by the competitive binding method with protein. It is shown that despite the possible differences in the concrete mechanism of action of the hypolipidemic agents chosen for study on the cyclase system, the use of suchmore » agents, offers definite prospects for oriented modification of the hormone sensitivity of tumor cells.« less

  12. Receptor-type guanylate cyclase is required for carbon dioxide sensation by Caenorhabditis elegans.

    PubMed

    Hallem, Elissa A; Spencer, W Clay; McWhirter, Rebecca D; Zeller, Georg; Henz, Stefan R; Rätsch, Gunnar; Miller, David M; Horvitz, H Robert; Sternberg, Paul W; Ringstad, Niels

    2011-01-04

    CO(2) is both a critical regulator of animal physiology and an important sensory cue for many animals for host detection, food location, and mate finding. The free-living soil nematode Caenorhabditis elegans shows CO(2) avoidance behavior, which requires a pair of ciliated sensory neurons, the BAG neurons. Using in vivo calcium imaging, we show that CO(2) specifically activates the BAG neurons and that the CO(2)-sensing function of BAG neurons requires TAX-2/TAX-4 cyclic nucleotide-gated ion channels and the receptor-type guanylate cyclase GCY-9. Our results delineate a molecular pathway for CO(2) sensing and suggest that activation of a receptor-type guanylate cyclase is an evolutionarily conserved mechanism by which animals detect environmental CO(2).

  13. Partial reactions of d-glucose 6-phosphate–1 l-myoinositol 1-phosphate cyclase

    PubMed Central

    Barnett, J. E. G.; Rasheed, A.; Corina, D. L.

    1973-01-01

    After removal of tightly bound NAD+ by using charcoal, a preparation of d-glucose 6-phosphate–1 l-myoinositol 1-phosphate cyclase catalysed the reduction of 5-keto-d-glucitol 6-phosphate and 5-keto-d-glucose 6-phosphate by [4-3H]NADH to give [5-3H]-glucitol 6-phosphate and [5-3H]glucose 6-phosphate respectively. The position of the tritium atom in the latter was shown by degradation. Both enzyme-catalysed reductions were strongly inhibited by 2-deoxy-d-glucose 6-phosphate, a powerful competitive inhibitor of inositol cyclase. The charcoal-treated enzyme preparation also converted 5-keto-d-glucose 6-phosphate into [3H]myoinositol 1-phosphate in the presence of [4-3H]NADH, but less effectively. These partial reactions of inositol cyclase are interpreted as providing strong evidence for the formation of 5-keto-d-glucose 6-phosphate as an enzyme-bound intermediate in the conversion of d-glucose 6-phosphate into 1 l-myoinositol 1-phosphate. The enzyme was partially inactivated by NaBH4 in the presence of NAD+. Glucose 6-phosphate did not increase the inactivation, and there was no inactivation in the absence of NAD+. There was no evidence for Schiff base formation during the cyclization. d-Glucitol 6-phosphate (l-sorbitol 1-phosphate) was a good inhibitor of the overall reaction. It did not inactivate the enzyme. The apparent molecular weight of inositol cyclase as determined by Sephadex chromatography was 2.15×105. PMID:4352864

  14. Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in Syrian hamster hypothalamus

    SciTech Connect

    Niles, L.P.; Hashemi, F.

    1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, ({sup 125}I)iodomelatonin, was examined using an incubation temperature (30{degree}C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing ({sup 125}I)iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax =more » 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus.« less

  15. The first structure of a bacterial diterpene cyclase: CotB2.

    PubMed

    Janke, Ronja; Görner, Christian; Hirte, Max; Brück, Thomas; Loll, Bernhard

    2014-06-01

    Sesquiterpenes and diterpenes are a diverse class of secondary metabolites that are predominantly derived from plants and some prokaryotes. The properties of these natural products encompass antitumor, antibiotic and even insecticidal activities. Therefore, they are interesting commercial targets for the chemical and pharmaceutical industries. Owing to their structural complexity, these compounds are more efficiently accessed by metabolic engineering of microbial systems than by chemical synthesis. This work presents the first crystal structure of a bacterial diterpene cyclase, CotB2 from the soil bacterium Streptomyces melanosporofaciens, at 1.64 Å resolution. CotB2 is a diterpene cyclase that catalyzes the cyclization of the linear geranylgeranyl diphosphate to the tricyclic cyclooctat-9-en-7-ol. The subsequent oxidation of cyclooctat-9-en-7-ol by two cytochrome P450 monooxygenases leads to bioactive cyclooctatin. Plasticity residues that decorate the active site of CotB2 have been mutated, resulting in alternative monocyclic, dicyclic and tricyclic compounds that show bioactivity. These new compounds shed new light on diterpene cyclase reaction mechanisms. Furthermore, the product of mutant CotB2(W288G) produced the new antibiotic compound (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene, which acts specifically against multidrug-resistant Staphylococcus aureus. This opens a sustainable route for the industrial-scale production of this bioactive compound.

  16. Crystallization and preliminary X-ray diffraction studies of the glutaminyl cyclase from Carica papaya latex

    SciTech Connect

    Azarkan, Mohamed; Clantin, Bernard; Bompard, Coralie

    2005-01-01

    The glutaminyl cyclase isolated from C. papaya latex has been crystallized using the hanging-drop method. Diffraction data have been collected at ESRF beamline BM14 and processed to 1.7 Å resolution. In living systems, the intramolecular cyclization of N-terminal glutamine residues is accomplished by glutaminyl cyclase enzymes (EC 2.3.2.5). While in mammals these enzymes are involved in the synthesis of hormonal and neurotransmitter peptides, the physiological role played by the corresponding plant enzymes still remains to be unravelled. Papaya glutaminyl cyclase (PQC), a 33 kDa enzyme found in the latex of the tropical tree Carica papaya, displays an exceptional resistance tomore » chemical and thermal denaturation as well as to proteolysis. In order to elucidate its enzymatic mechanism and to gain insights into the structural determinants underlying its remarkable stability, PQC was isolated from papaya latex, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 62.82, b = 81.23, c = 108.17 Å and two molecules per asymmetric unit. Diffraction data have been collected at ESRF beamline BM14 and processed to a resolution of 1.7 Å.« less

  17. The Presence of Two Cyclase Thioesterases Expands the Conformational Freedom of the Cyclic Peptide Occidiofungin

    PubMed Central

    Ravichandran, Akshaya; Gu, Ganyu; Escano, Jerome; Lu, Shi-En; Smith, Leif

    2014-01-01

    Occidiofungin is a cyclic nonribosomally synthesized antifungal peptide with submicromolar activity produced by Gram-negative bacterium Burkholderia contaminans. The biosynthetic gene cluster was confirmed to contain two cyclase thioesterases. NMR analysis revealed that the presence of both thioesterases is used to increase the conformational repertoire of the cyclic peptide. The loss of the OcfN cyclic thioesterase by mutagenesis results in a reduction of conformational variants and an appreciable decrease in bioactivity against Candida species. Presumably, the presence of both asparagine and β-hydroxyasparagine variants coordinate the enzymatic function of both of the cyclase thioesterases. OcfN has presumably evolved to be part of the biosynthetic gene cluster due to its ability to produce structural variants that enhance antifungal activity against some fungi. The enhancement of the antifungal activity from the incorporation of an additional cyclase thioesterase into the biosynthetic gene cluster of occidiofungin supports the need to explore new conformational variants of other therapeutic or potentially therapeutic cyclic peptides. PMID:23394257

  18. Prostaglandin E2 Stimulates EP2, Adenylate Cyclase, Phospholipase C, and Intracellular Calcium Release to Mediate Cyclic Adenosine Monophosphate Production in Dental Pulp Cells.

    PubMed

    Chang, Mei-Chi; Lin, Szu-I; Lin, Li-Deh; Chan, Chiu-Po; Lee, Ming-Shu; Wang, Tong-Mei; Jeng, Po-Yuan; Yeung, Sin-Yuet; Jeng, Jiiang-Huei

    2016-04-01

    Prostaglandin E2 (PGE2) plays a crucial role in pulpal inflammation and repair. However, its induction of signal transduction pathways is not clear but is crucial for future control of pulpal inflammation. Primary dental pulp cells were exposed to PGE2 and 19R-OH PGE2 (EP2 agonist) or sulprostone (EP1/EP3 agonist) for 5 to 40 minutes. Cellular cyclic adenosine monophosphate (cAMP) levels were measured using the enzyme-linked immunosorbent assay. In some experiments, cells were pretreated with SQ22536 (adenylate cyclase inhibitor), H89 (protein kinase A inhibitor), dorsomorphin (adenosine monophosphate-activated protein kinase inhibitor), U73122 (phospholipase C inhibitor), thapsigargin (inhibitor of intracellular calcium release), W7 (calmodulin antagonist), verapamil (L-type calcium channel blocker), and EGTA (extracellular calcium chelator) for 20 minutes before the addition of PGE2. PGE2 and 19R-OH PGE2 (EP2 agonist) stimulated cAMP production, whereas sulprostone (EP1/EP3 agonist) shows little effect. PGE2-induced cAMP production was attenuated by SQ22536 and U73122 but not H89 and dorsomorphin. Intriguingly, thapsigargin and W7 prevented PGE2-induced cAMP production, but verapamil and EGTA showed little effect. These results indicate that PGE2-induced cAMP production is associated with EP2 receptor and adenylate cyclase activation. These events are mediated by phospholipase C, intracellular calcium release, and calcium-calmodulin signaling. These results are helpful for understanding the role of PGE2 in pulpal inflammation and repair and possible future drug intervention. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  19. Short-term hyperthyroidism modulates adenosine receptors and catalytic activity of adenylate cyclase in adipocytes.

    PubMed Central

    Rapiejko, P J; Malbon, C C

    1987-01-01

    The effects of short-term hyperthyroidism in vivo on the status of the components of the fat-cell hormone-sensitive adenylate cyclase were investigated. The number of beta-adrenergic receptors was elevated by about 25% in membranes of fat-cells isolated from hyperthyroid rats as compared with euthyroid rats, but their affinity for radioligand was unchanged. Membranes of hyperthyroid-rat fat-cells displayed less than 65% of the normal complement of receptors for [3H]cyclohexyladenosine. The affinity of the receptors for this ligand was normal. In contrast with the marked increase in the amounts of the alpha-subunits of the guanine nucleotide-binding proteins Gi (Mr 41,000) and Go (Mr 39,000) observed in the hypothyroid state [Malbon, Rapiejko & Mangano (1985) J. Biol. Chem. 260, 2558-2564], the amounts of alpha-Gi, alpha-Go as well as alpha-Gs subunits [Mr 42,000 (major) and 46,000/48,000 (minor)] were not changed by hyperthyroidism. Adenylate cyclase activity in response to forskolin, guanosine 5'-[gamma-thio]triphosphate or isoprenaline, in contrast, was decreased by 30-50% in fat-cell membranes from hyperthyroid rats. Fat-cells isolated from hyperthyroid rats accumulated cyclic AMP to less than 50% of the extent in their euthyroid counterparts in the presence of adenosine deaminase and either adrenaline or forskolin, suggesting a decrease in the amount or activity of the catalytic subunit of adenylate cyclase. In the absence of exogenous adenosine deaminase, cyclic AMP accumulation in response to adrenaline was elevated rather than decreased in fat-cells from hyperthyroid rats. The inhibitory influence of adenosine is apparently limited in the hyperthyroid state by the decreased complement of inhibitory R-site purinergic receptors in these fat-cells. Short-term hyperthyroidism modulates the fat-cell adenylate cyclase system at the receptor level (beta-receptor number increased, R-site purinergic-receptor number decreased) and the catalytic subunit of adenylate

  20. Effects of the NO/soluble guanylate cyclase/cGMP system on the functions of human platelets.

    PubMed

    Makhoul, Stephanie; Walter, Elena; Pagel, Oliver; Walter, Ulrich; Sickmann, Albert; Gambaryan, Stepan; Smolenski, Albert; Zahedi, René P; Jurk, Kerstin

    2018-06-01

    Platelets are circulating sentinels of vascular integrity and are activated, inhibited, or modulated by multiple hormones, vasoactive substances or drugs. Endothelium- or drug-derived NO strongly inhibits platelet activation via activation of the soluble guanylate cyclase (sGC) and cGMP elevation, often in synergy with cAMP-elevation by prostacyclin. However, the molecular mechanisms and diversity of cGMP effects in platelets are poorly understood and sometimes controversial. Recently, we established the quantitative human platelet proteome, the iloprost/prostacyclin/cAMP/protein kinase A (PKA)-regulated phosphoproteome, and the interactions of the ADP- and iloprost/prostacyclin-affected phosphoproteome. We also showed that the sGC stimulator riociguat is in vitro a highly specific inhibitor, via cGMP, of various functions of human platelets. Here, we review the regulatory role of the cGMP/protein kinase G (PKG) system in human platelet function, and our current approaches to establish and analyze the phosphoproteome after selective stimulation of the sGC/cGMP pathway by NO donors and riociguat. Present data indicate an extensive and diverse NO/riociguat/cGMP phosphoproteome, which has to be compared with the cAMP phosphoproteome. In particular, sGC/cGMP-regulated phosphorylation of many membrane proteins, G-proteins and their regulators, signaling molecules, protein kinases, and proteins involved in Ca 2+ regulation, suggests that the sGC/cGMP system targets multiple signaling networks rather than a limited number of PKG substrate proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. Sphingosine 1-phosphate stimulates hydrogen peroxide generation through activation of phospholipase C-Ca2+ system in FRTL-5 thyroid cells: possible involvement of guanosine triphosphate-binding proteins in the lipid signaling.

    PubMed

    Okajima, F; Tomura, H; Sho, K; Kimura, T; Sato, K; Im, D S; Akbar, M; Kondo, Y

    1997-01-01

    Exogenous sphingosine 1-phosphate (S1P) stimulated hydrogen peroxide (H2O2) generation in association with an increase in intracellular Ca2+ concentration in FRTL-5 thyroid cells. S1P also induced inositol phosphate production, reflecting activation of phospholipase C (PLC) in the cells. These three S1P-induced events were inhibited partially by pertussis toxin (PTX) and markedly by U73122, a PLC inhibitor, and were conversely potentiated by N6-(L-2-phenylisopropyl)adenosine, an A1-adenosine receptor agonist. In FRTL-5 cell membranes, S1P also activated PLC in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), but not in its absence. Guanosine 5'-O-(2-thiodiphosphate) inhibited the S1P-induced GTP gamma S-dependent activation of the enzyme. To characterize the signaling pathways, especially receptors and G proteins involved in the S1P-induced responses, cross-desensitization experiments were performed. Under the conditions where homologous desensitization occurred in S1P-, lysophosphatidic acid (LPA)-, and bradykinin-induced induction of Ca2+ mobilization, no detectable cross-desensitization of S1P and bradykinin was observed. This suggests that the primary action of S1P in its activation of the PLC-Ca2+ system was not the activation of G proteins common to S1P and bradykinin, but the activation of a putative S1P receptor. On the other hand, there was a significant cross-desensitization of S1P and LPA; however, a still significant response to S1P (50-80% of the response in the nontreated control cells) was observed depending on the lipid dose employed after a prior LPA challenge. S1P also inhibited cAMP accumulation in a PTX-sensitive manner. We conclude that S1P stimulates H2O2 generation through a PLC-Ca2+ system and also inhibits adenylyl cyclase in FRTL-5 thyroid cells. The S1P-induced responses may be mediated partly through a putative lipid receptor that is coupled to both PTX-sensitive and insensitive G proteins.

  2. Pituitary adenylate cyclase activating polypeptide (PACAP), stress, and sex hormones.

    PubMed

    King, S Bradley; Toufexis, Donna J; Hammack, Sayamwong E

    2017-09-01

    Stressor exposure is associated with the onset and severity of many psychopathologies that are more common in women than men. Moreover, the maladaptive expression and function of stress-related hormones have been implicated in these disorders. Evidence suggests that PACAP has a critical role in the stress circuits mediating stress-responding, and PACAP may interact with sex hormones to contribute to sex differences in stress-related disease. In this review, we describe the role of the PACAP/PAC1 system in stress biology, focusing on the role of stress-induced alterations in PACAP expression and signaling in the development of stress-induced behavioral change. Additionally, we present more recent data suggesting potential interactions between stress, PACAP, and circulating estradiol in pathological states, including PTSD. These studies suggest that the level of stress and circulating gonadal hormones may differentially regulate the PACAPergic system in males and females to influence anxiety-like behavior and may be one mechanism underlying the discrepancies in human psychiatric disorders.

  3. Intestinal receptor for heat-stable enterotoxin of Escherichia coli is tightly coupled to a novel form of particulate guanylate cyclase.

    PubMed Central

    Waldman, S A; Kuno, T; Kamisaki, Y; Chang, L Y; Gariepy, J; O'Hanley, P; Schoolnik, G; Murad, F

    1986-01-01

    A novel form of particulate guanylate cyclase tightly coupled by cytoskeletal components to receptors for heat-stable enterotoxin (ST) produced by Escherichia coli can be found in membranes from rat intestinal mucosa. Intestinal particulate guanylate cyclase was resistant to solubilization with detergent alone, with only 30% of the total enzyme activity being extracted with Lubrol-PX. Under similar conditions, 70% of this enzyme was solubilized from rat lung membranes. The addition of high concentrations of sodium chloride to the extraction buffer resulted in greater solubilization of particulate guanylate cyclase from intestinal membranes. Although extraction of intestinal membranes with detergent and salt resulted in greater solubilization of guanylate cyclase, a small fraction of the enzyme activity remained associated with the particulate fraction. This activity was completely resistant to solubilization with a variety of detergents and chaotropes. Particulate guanylate cyclase and the ST receptor solubilized by detergent retained their abilities to produce cyclic GMP and bind ST, respectively. However, ST failed to activate particulate guanylate cyclase in detergent extracts. In contrast, guanylate cyclase resistant to solubilization remained functional and coupled to the ST receptor since enzyme activation by ST was unaffected by various extraction procedures. The possibility that the ST receptor and particulate guanylate cyclase were the same molecule was explored. ST binding and cyclic GMP production were separated by affinity chromatography on GTP-agarose. Similarly, guanylate cyclase migrated as a 300,000-dalton protein, while the ST receptor migrated as a 240,000-dalton protein on gel filtration chromatography. Also, thiol-reactive agents such as cystamine and N-ethylmaleimide inhibited guanylate cyclase activation by ST, with no effect on receptor binding of ST. These data suggest that guanylate cyclase and the ST receptor are independent proteins

  4. Rationale and design of the SOluble guanylate Cyclase stimulatoR in heArT failurE Studies (SOCRATES).

    PubMed

    Pieske, Burkert; Butler, Javed; Filippatos, Gerasimos; Lam, Carolyn; Maggioni, Aldo Pietro; Ponikowski, Piotr; Shah, Sanjiv; Solomon, Scott; Kraigher-Krainer, Elisabeth; Samano, Eliana Tibana; Scalise, Andrea Viviana; Müller, Katharina; Roessig, Lothar; Gheorghiade, Mihai

    2014-09-01

    The clinical outcomes for patients with worsening chronic heart failure (WCHF) remain exceedingly poor despite contemporary evidence-based therapies, and effective therapies are urgently needed. Accumulating evidence supports augmentation of cyclic guanosine monophosphate (cGMP) signalling as a potential therapeutic strategy for HF with reduced or preserved ejection fraction (HFrEF and HFpEF, respectively). Direct soluble guanylate cyclase (sGC) stimulators target reduced cGMP generation due to insufficient sGC stimulation and represent a promising method for cGMP enhancement. The phase II SOluble guanylate Cyclase stimulatoR in heArT failurE Study (SOCRATES) programme consists of two randomized, parallel-group, placebo-controlled, double-blind, multicentre studies, SOCRATES-REDUCED (in patients with LVEF <45%) and SOCRATES-PRESERVED (in those with LVEF ≥ 45%), that will explore the pharmacodynamic effects, safety and tolerability, and pharmacokinetics of four dose regimens of the once-daily oral sGC stimulator vericiguat (BAY 1021189) over 12 weeks compared with placebo. These studies will enrol patients stabilized during hospitalization for HF at the time of discharge or within 4 weeks thereafter. The primary endpoint in SOCRATES-REDUCED is change in NT-proBNP at 12 weeks. The primary endpoints in SOCRATES-PRESERVED are change in NT-proBNP and left atrial volume at 12 weeks. SOCRATES will be the first programme to enrol specifically both inpatients and outpatients with WCHF and patients with reduced or preserved ejection fraction. Results will inform the benefits of pursuing subsequent event-driven clinical outcome trials with sGC stimulators in this patient population. © 2014 The Authors. European Journal of Heart Failure © 2014 European Society of Cardiology.

  5. Unifying mechanism for Aplysia ADP-ribosyl cyclase and CD38/NAD(+) glycohydrolases.

    PubMed Central

    Cakir-Kiefer, C; Muller-Steffner, H; Schuber, F

    2000-01-01

    Highly purified Aplysia californica ADP-ribosyl cyclase was found to be a multifunctional enzyme. In addition to the known transformation of NAD(+) into cADP-ribose this enzyme is able to catalyse the solvolysis (hydrolysis and methanolysis) of cADP-ribose. This cADP-ribose hydrolase activity, which becomes detectable only at high concentrations of the enzyme, is amplified with analogues such as pyridine adenine dinucleotide, in which the cleavage rate of the pyridinium-ribose bond is much reduced compared with NAD(+). Although the specificity ratio V(max)/K(m) is in favour of NAD(+) by 4 orders of magnitude, this multifunctionality allowed us to propose a 'partitioning' reaction scheme for the Aplysia enzyme, similar to that established previously for mammalian CD38/NAD(+) glycohydrolases. This mechanism involves the formation of a single oxocarbenium-type intermediate that partitions to cADP-ribose and solvolytic products via competing pathways. In favour of this mechanism was the finding that the enzyme also catalysed the hydrolysis of NMN(+), a substrate that cannot undergo cyclization. The major difference between the mammalian and the invertebrate enzymes resides in their relative cyclization/hydrolysis rate-constant ratios, which dictate their respective yields of cADP-ribose (ADP-ribosyl cyclase activity) and ADP-ribose (NAD(+) glycohydrolase activity). For the Aplysia enzyme's catalysed transformation of NAD(+) we favour a mechanism where the formation of cADP-ribose precedes that of ADP-ribose; i.e. macroscopically the invertebrate ADP-ribosyl cyclase conforms to a sequential reaction pathway as a limiting form of the partitioning mechanism. PMID:10861229

  6. [Construction of high-yield strain by optimizing lycopene cyclase for β-carotene production].

    PubMed

    Jin, Yingfu; Han, Li; Zhang, Shasha; Li, Shizhong; Liu, Weifeng; Tao, Yong

    2017-11-25

    To optimize key enzymes, such as to explore the gene resources and to modify the expression level, can maximize metabolic pathways of target products. β-carotene is a terpenoid compound with important application value. Lycopene cyclase (CrtY) is the key enzyme in β-carotene biosynthesis pathway, catalyzing flavin adenine dinucleotide (FAD)-dependent cyclization reaction and β-carotene synthesis from lycopene precursor. We optimized lycopene cyclase (CrtY) to improve the synthesis of β-carotene and determined the effect of CrtY expression on metabolic pathways. Frist, we developed a β-carotene synthesis module by coexpressing the lycopene β-cyclase gene crtY with crtEBI module in Escherichia coli. Then we simultaneously optimized the ribosome-binding site (RBS) intensity and the species of crtY using oligo-linker mediated DNA assembly method (OLMA). Five strains with high β-carotene production capacity were screened out from the OLMA library. The β-carotene yields of these strains were up to 15.79-18.90 mg/g DCW (Dry cell weight), 65% higher than that of the original strain at shake flask level. The optimal strain CP12 was further identified and evaluated for β-carotene production at 5 L fermentation level. After process optimization, the final β-carotene yield could reach to 1.9 g/L. The results of RBS strength and metabolic intermediate analysis indicated that an appropriate expression level of CrtY could be beneficial for the function of the β-carotene synthesis module. The results of this study provide important insight into the optimization of β-carotene synthesis pathway in metabolic engineering.

  7. Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes

    DOE PAGES

    Wu, Vincent W.; Dana, Craig M.; Iavarone, Anthony T.; ...

    2017-01-17

    The breakdown of plant biomass to simple sugars is essential for the production of second-generation biofuels and high-value bioproducts. Currently, enzymes produced from filamentous fungi are used for deconstructing plant cell wall polysaccharides into fermentable sugars for biorefinery applications. A post-translational N-terminal pyroglutamate modification observed in some of these enzymes occurs when N-terminal glutamine or glutamate is cyclized to form a five-membered ring. This modification has been shown to confer resistance to thermal denaturation for CBH-1 and EG-1 cellulases. In mammalian cells, the formation of pyroglutamate is catalyzed by glutaminyl cyclases. Using the model filamentous fungus Neurospora crassa, we identifiedmore » two genes ( qc-1 and qc-2) that encode proteins homologous to mammalian glutaminyl cyclases. We show that qc-1 and qc-2 are essential for catalyzing the formation of an N-terminal pyroglutamate on CBH-1 and GH5-1. CBH-1 and GH5-1 produced in a Δqc-1 Δqc-2 mutant, and thus lacking the N-terminal pyroglutamate modification, showed greater sensitivity to thermal denaturation, and for GH5-1, susceptibility to proteolytic cleavage. QC-1 and QC-2 are endoplasmic reticulum (ER)-localized proteins. The pyroglutamate modification is predicted to occur in a number of additional fungal proteins that have diverse functions. The identification of glutaminyl cyclases in fungi may have implications for production of lignocellulolytic enzymes, heterologous expression, and biotechnological applications revolving around protein stability.« less

  8. Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes

    SciTech Connect

    Wu, Vincent W.; Dana, Craig M.; Iavarone, Anthony T.

    The breakdown of plant biomass to simple sugars is essential for the production of second-generation biofuels and high-value bioproducts. Currently, enzymes produced from filamentous fungi are used for deconstructing plant cell wall polysaccharides into fermentable sugars for biorefinery applications. A post-translational N-terminal pyroglutamate modification observed in some of these enzymes occurs when N-terminal glutamine or glutamate is cyclized to form a five-membered ring. This modification has been shown to confer resistance to thermal denaturation for CBH-1 and EG-1 cellulases. In mammalian cells, the formation of pyroglutamate is catalyzed by glutaminyl cyclases. Using the model filamentous fungus Neurospora crassa, we identifiedmore » two genes ( qc-1 and qc-2) that encode proteins homologous to mammalian glutaminyl cyclases. We show that qc-1 and qc-2 are essential for catalyzing the formation of an N-terminal pyroglutamate on CBH-1 and GH5-1. CBH-1 and GH5-1 produced in a Δqc-1 Δqc-2 mutant, and thus lacking the N-terminal pyroglutamate modification, showed greater sensitivity to thermal denaturation, and for GH5-1, susceptibility to proteolytic cleavage. QC-1 and QC-2 are endoplasmic reticulum (ER)-localized proteins. The pyroglutamate modification is predicted to occur in a number of additional fungal proteins that have diverse functions. The identification of glutaminyl cyclases in fungi may have implications for production of lignocellulolytic enzymes, heterologous expression, and biotechnological applications revolving around protein stability.« less

  9. Regulation and therapeutic targeting of peptide-activated receptor guanylyl cyclases

    PubMed Central

    Potter, Lincoln R.

    2016-01-01

    Cyclic GMP is a ubiquitous second messenger that regulates a wide array of physiologic processes such as blood pressure, long bone growth, intestinal fluid secretion, phototransduction and lipolysis. Soluble and single-membrane-spanning enzymes called guanylyl cyclases (GC) synthesize cGMP. In humans, the latter group consists of GC-A, GC-B, GC-C, GC-E and GC-F, which are also known as NPR-A, NPR-B, StaR, Ret1-GC and Ret2-GC, respectively. Membrane GCs are activated by peptide ligands such as atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), C-type natriuretic peptide (CNP), guanylin, uroguanylin, heat stable enterotoxin and GC-activating proteins. Nesiritide and carperitide are clinically approved peptide-based drugs that activate GC-A. CD-NP is an experimental heart failure drug that primarily activates GC-B but also activates GC-A at high concentrations and is resistant to degradation. Inactivating mutations in GC-B cause acromesomelic dysplasia type Maroteaux dwarfism and chromosomal mutations that increase CNP concentrations are associated with Marfanoid-like skeletal overgrowth. Pump-based CNP infusions increase skeletal growth in a mouse model of the most common type of human dwarfism, which supports CNP/GC-B-based therapies for short stature diseases. Linaclotide is a peptide activator of GC-C that stimulates intestinal motility and is in late-stage clinical trials for the treatment of chronic constipation. This review discusses the discovery of cGMP, guanylyl cyclases, the general characteristics and therapeutic applications of GC-A, GC-B and GC-C, and emphasizes the regulation of transmembrane guanylyl cyclases by phosphorylation and ATP. PMID:21185863

  10. Multifunctional oxidosqualene cyclases and cytochrome P450 involved in the biosynthesis of apple fruit triterpenic acids.

    PubMed

    Andre, Christelle M; Legay, Sylvain; Deleruelle, Amélie; Nieuwenhuizen, Niels; Punter, Matthew; Brendolise, Cyril; Cooney, Janine M; Lateur, Marc; Hausman, Jean-François; Larondelle, Yvan; Laing, William A

    2016-09-01

    Apple (Malus × domestica) accumulates bioactive ursane-, oleanane-, and lupane-type triterpenes in its fruit cuticle, but their biosynthetic pathway is still poorly understood. We used a homology-based approach to identify and functionally characterize two new oxidosqualene cyclases (MdOSC4 and MdOSC5) and one cytochrome P450 (CYP716A175). The gene expression patterns of these enzymes and of previously described oxidosqualene cyclases were further studied in 20 apple cultivars with contrasting triterpene profiles. MdOSC4 encodes a multifunctional oxidosqualene cyclase producing an oleanane-type triterpene, putatively identified as germanicol, as well as β-amyrin and lupeol, in the proportion 82 : 14 : 4. MdOSC5 cyclizes 2,3-oxidosqualene into lupeol and β-amyrin at a ratio of 95 : 5. CYP716A175 catalyses the C-28 oxidation of α-amyrin, β-amyrin, lupeol and germanicol, producing ursolic acid, oleanolic acid, betulinic acid, and putatively morolic acid. The gene expression of MdOSC1 was linked to the concentrations of ursolic and oleanolic acid, whereas the expression of MdOSC5 was correlated with the concentrations of betulinic acid and its caffeate derivatives. Two new multifuntional triterpene synthases as well as a multifunctional triterpene C-28 oxidase were identified in Malus × domestica. This study also suggests that MdOSC1 and MdOSC5 are key genes in apple fruit triterpene biosynthesis. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  11. Developing grasshopper neurons show variable levels of guanylyl cyclase activity on arrival at their targets.

    PubMed

    Ball, E E; Truman, J W

    1998-04-27

    The ability of certain grasshopper neurons to respond to exogenously applied donors of nitric oxide (NO) by producing cyclic GMP (cGMP) depends on their developmental state. ODQ, a selective blocker of NO-sensitive guanylyl cyclase, blocks cGMP production at 10(-5) M, thus confirming the nature of the response. Experiments in which the distal axon is separated from its proximal stump before application of an NO donor show that guanylyl cyclase is distributed uniformly throughout the neuron. In the locust abdomen, where segments are formed sequentially, the pattern of guanylyl cyclase up-regulation is predictable and sequential from anterior to posterior. There are two patterns of innervation by cGMP-expressing motor neurons. In the first, typified by muscle 187, an innervating neuron begins to be NO responsive on arrival at its muscle and continues to be so over most of the remainder of embryonic development, including the formation of motor end plates. In the second, typified by a neuron innervating muscle 191, the neuron extends well along the muscle, apparently laying down a number of sites of contact with it, before it becomes NO responsive. In both patterns, however, NO responsiveness marks the neuron's transition from growth cone elongation to the production of lateral branches. Individual muscles receive innervation from multiple motor neurons, some of which express transient NO sensitivity during development and others which do not. With the exception of the leg motor neuron SETi, the first motor neuron to reach any muscle is usually not NO responsive. We suggest that cGMP plays a role in, or reflects, the early stages of communication between a target and specific innervating neurons.

  12. Differential effects of non-ionic detergents on microsomal and sarcolemmal adenylate cyclase in cardiac muscle

    PubMed Central

    Sulakhe, Prakash V.; Narayanan, Njanoor

    1978-01-01

    1. About 4 and 23% of the homogenate adenylate cyclase activity was recovered in the microsomal and sarcolemmal fractions isolated from guinea-pig heart ventricles. 2. Cardiac microsomal adenylate cyclase activity [basal as well as p[NH]ppG (guanyl-5′-yl imidodiphosphate)- and NaF-stimulated] was increased over 2-fold in the presence of Lubrol-PX (0.01–0.1%). 3. The sarcolemmal enzyme, however, showed concentration-dependent inhibition caused by the detergent under all assay conditions, except when p[NH]ppG was included in the assay. In the latter case, the detergent (0.01–0.02%) caused a modest increase (30–45%) in enzyme activity. 4. Another non-ionic detergent, Triton X-100, also stimulated the microsomal cyclase and inhibited the sarcolemmal enzyme. 5. With either membrane fraction, Lubrol-PX solubilized the enzyme when the detergent/membrane protein ratio was 2.5 (μmol of detergent/mg of protein). 6. The findings with homogenate and a washed particulate fraction resembled those obtained with sarcolemma, and those with isolated sarcoplasmic reticulum resembled those with microsomal preparations. 7. p[NH]ppG, and to some extent NaF, protected the detergent-induced inactivation of the enzyme observed at higher detergent concentrations (0.5% Lubrol-PX and 0.05–0.5% Triton X-100). 8. In the absence of detergents, p[NH]ppG increased the basal enzyme activity about 2-fold in microsomal fractions, but did not appreciably stimulate the sarcolemmal enzyme. Isoproterenol, on the other hand, increased the sarcolemmal enzyme activity (>2-fold) in the presence of p[NH]ppG and caused only moderate stimulation (31%) of the microsomal enzyme under these conditions. 9. These findings support the view that, although the bulk of adenylate cyclase resides in heart sarcolemma (plasma membrane), the microsomal activity cannot be accounted for solely by contamination of the microsomal fraction with sarcolemma, as has been suggested by others [Besch, Jones & Watanabe (1976

  13. Structural basis for olivetolic acid formation by a polyketide cyclase from Cannabis sativa.

    PubMed

    Yang, Xinmei; Matsui, Takashi; Kodama, Takeshi; Mori, Takahiro; Zhou, Xiaoxi; Taura, Futoshi; Noguchi, Hiroshi; Abe, Ikuro; Morita, Hiroyuki

    2016-03-01

    In polyketide biosynthesis, ring formation is one of the key diversification steps. Olivetolic acid cyclase (OAC) from Cannabis sativa, involved in cannabinoid biosynthesis, is the only known plant polyketide cyclase. In addition, it is the only functionally characterized plant α+β barrel (DABB) protein that catalyzes the C2-C7 aldol cyclization of the linear pentyl tetra-β-ketide CoA as the substrate, to generate olivetolic acid (OA). Herein, we solved the OAC apo and OAC-OA complex binary crystal structures at 1.32 and 1.70 Å resolutions, respectively. The crystal structures revealed that the enzyme indeed belongs to the DABB superfamily, as previously proposed, and possesses a unique active-site cavity containing the pentyl-binding hydrophobic pocket and the polyketide binding site, which have never been observed among the functionally and structurally characterized bacterial polyketide cyclases. Furthermore, site-directed mutagenesis studies indicated that Tyr72 and His78 function as acid/base catalysts at the catalytic center. Structural and/or functional studies of OAC suggested that the enzyme lacks thioesterase and aromatase activities. These observations demonstrated that OAC employs unique catalytic machinery utilizing acid/base catalytic chemistry for the formation of the precursor of OA. The structural and functional insights obtained in this work thus provide the foundation for analyses of the plant polyketide cyclases that will be discovered in the future. Structural data reported in this paper are available in the Protein Data Bank under the accession numbers 5B08 for the OAC apo, 5B09 for the OAC-OA binary complex and 5B0A, 5B0B, 5B0C, 5B0D, 5B0E, 5B0F and 5B0G for the OAC His5Q, Ile7F, Tyr27F, Tyr27W, Val59M, Tyr72F and His78S mutant enzymes, respectively. © 2016 Federation of European Biochemical Societies.

  14. The fibrate gemfibrozil is a NO- and haem-independent activator of soluble guanylyl cyclase: in vitro studies

    PubMed Central

    Sharina, I G; Sobolevsky, M; Papakyriakou, A; Rukoyatkina, N; Spyroulias, G A; Gambaryan, S; Martin, E

    2015-01-01

    Background and Purpose Fibrates are a class of drugs widely used to treat dyslipidaemias. They regulate lipid metabolism and act as PPARα agonists. Clinical trials demonstrate that besides changes in lipid profiles, fibrates decrease the incidence of cardiovascular events, with gemfibrozil exhibiting the most pronounced benefit. This study aims to characterize the effect of gemfibrozil on the activity and function of soluble guanylyl cyclase (sGC), the key mediator of NO signalling. Experimental Approach High-throughput screening of a drug library identified gemfibrozil as a direct sGC activator. Activation of sGC is unique to gemfibrozil and is not shared by other fibrates. Key Results Gemfibrozil activated purified sGC, induced endothelium-independent relaxation of aortic rings and inhibited platelet aggregation. Gemfibrozil-dependent activation was absent when the sGC haem domain was deleted, but was significantly enhanced when sGC haem was lacking or oxidized. Oxidation of sGC haem enhanced the vasoactive and anti-platelet effects of gemfibrozil. Gemfibrozil competed with the haem-independent sGC activators ataciguat and cinaciguat. Computational modelling predicted that gemfibrozil occupies the space of the haem group and interacts with residues crucial for haem stabilization. This is consistent with structure-activity data which revealed an absolute requirement for gemfibrozil's carboxyl group. Conclusions and Implications These data suggest that in addition to altered lipid and lipoprotein state, the cardiovascular preventive benefits of gemfibrozil may derive from direct activation and protection of sGC function. A sGC-directed action may explain the more pronounced cardiovascular benefit of gemfibrozil observed over other fibrates and some of the described side effects of gemfibrozil. PMID:25536881

  15. The fibrate gemfibrozil is a NO- and haem-independent activator of soluble guanylyl cyclase: in vitro studies.

    PubMed

    Sharina, I G; Sobolevsky, M; Papakyriakou, A; Rukoyatkina, N; Spyroulias, G A; Gambaryan, S; Martin, E

    2015-05-01

    Fibrates are a class of drugs widely used to treat dyslipidaemias. They regulate lipid metabolism and act as PPARα agonists. Clinical trials demonstrate that besides changes in lipid profiles, fibrates decrease the incidence of cardiovascular events, with gemfibrozil exhibiting the most pronounced benefit. This study aims to characterize the effect of gemfibrozil on the activity and function of soluble guanylyl cyclase (sGC), the key mediator of NO signalling. High-throughput screening of a drug library identified gemfibrozil as a direct sGC activator. Activation of sGC is unique to gemfibrozil and is not shared by other fibrates. Gemfibrozil activated purified sGC, induced endothelium-independent relaxation of aortic rings and inhibited platelet aggregation. Gemfibrozil-dependent activation was absent when the sGC haem domain was deleted, but was significantly enhanced when sGC haem was lacking or oxidized. Oxidation of sGC haem enhanced the vasoactive and anti-platelet effects of gemfibrozil. Gemfibrozil competed with the haem-independent sGC activators ataciguat and cinaciguat. Computational modelling predicted that gemfibrozil occupies the space of the haem group and interacts with residues crucial for haem stabilization. This is consistent with structure-activity data which revealed an absolute requirement for gemfibrozil's carboxyl group. These data suggest that in addition to altered lipid and lipoprotein state, the cardiovascular preventive benefits of gemfibrozil may derive from direct activation and protection of sGC function. A sGC-directed action may explain the more pronounced cardiovascular benefit of gemfibrozil observed over other fibrates and some of the described side effects of gemfibrozil. © 2014 The British Pharmacological Society.

  16. Natriuretic Peptide Receptor Guanylyl Cyclase-A in Podocytes is Renoprotective but Dispensable for Physiologic Renal Function.

    PubMed

    Staffel, Janina; Valletta, Daniela; Federlein, Anna; Ehm, Katharina; Volkmann, Regine; Füchsl, Andrea M; Witzgall, Ralph; Kuhn, Michaela; Schweda, Frank

    2017-01-01

    The cardiac natriuretic peptides (NPs), atrial NP and B-type NP, regulate fluid homeostasis and arterial BP through renal actions involving increased GFR and vascular and tubular effects. Guanylyl cyclase-A (GC-A), the transmembrane cGMP-producing receptor shared by these peptides, is expressed in different renal cell types, including podocytes, where its function is unclear. To study the effects of NPs on podocytes, we generated mice with a podocyte-specific knockout of GC-A (Podo-GC-A KO). Despite the marked reduction of GC-A mRNA in GC-A KO podocytes to 1% of the control level, Podo-GC-A KO mice and control littermates did not differ in BP, GFR, or natriuresis under baseline conditions. Moreover, infusion of synthetic NPs similarly increased the GFR and renal perfusion in both genotypes. Administration of the mineralocorticoid deoxycorticosterone-acetate (DOCA) in combination with high salt intake induced arterial hypertension of similar magnitude in Podo-GC-A KO mice and controls. However, only Podo-GC-A KO mice developed massive albuminuria (controls: 35-fold; KO: 5400-fold versus baseline), hypoalbuminemia, reduced GFR, and marked glomerular damage. Furthermore, DOCA treatment led to decreased expression of the slit diaphragm-associated proteins podocin, nephrin, and synaptopodin and to enhanced transient receptor potential canonical 6 (TRPC6) channel expression and ATP-induced calcium influx in podocytes of Podo-GC-A KO mice. Concomitant treatment of Podo-GC-A KO mice with the TRPC channel blocker SKF96365 markedly ameliorated albuminuria and glomerular damage in response to DOCA. In conclusion, the physiologic effects of NPs on GFR and natriuresis do not involve podocytes. However, NP/GC-A/cGMP signaling protects podocyte integrity under pathologic conditions, most likely by suppression of TRPC channels. Copyright © 2016 by the American Society of Nephrology.

  17. Natriuretic Peptide Receptor Guanylyl Cyclase-A in Podocytes is Renoprotective but Dispensable for Physiologic Renal Function

    PubMed Central

    Staffel, Janina; Valletta, Daniela; Federlein, Anna; Ehm, Katharina; Volkmann, Regine; Füchsl, Andrea M.; Witzgall, Ralph; Kuhn, Michaela

    2017-01-01

    The cardiac natriuretic peptides (NPs), atrial NP and B-type NP, regulate fluid homeostasis and arterial BP through renal actions involving increased GFR and vascular and tubular effects. Guanylyl cyclase-A (GC-A), the transmembrane cGMP-producing receptor shared by these peptides, is expressed in different renal cell types, including podocytes, where its function is unclear. To study the effects of NPs on podocytes, we generated mice with a podocyte-specific knockout of GC-A (Podo-GC-A KO). Despite the marked reduction of GC-A mRNA in GC-A KO podocytes to 1% of the control level, Podo-GC-A KO mice and control littermates did not differ in BP, GFR, or natriuresis under baseline conditions. Moreover, infusion of synthetic NPs similarly increased the GFR and renal perfusion in both genotypes. Administration of the mineralocorticoid deoxycorticosterone-acetate (DOCA) in combination with high salt intake induced arterial hypertension of similar magnitude in Podo-GC-A KO mice and controls. However, only Podo-GC-A KO mice developed massive albuminuria (controls: 35-fold; KO: 5400-fold versus baseline), hypoalbuminemia, reduced GFR, and marked glomerular damage. Furthermore, DOCA treatment led to decreased expression of the slit diaphragm-associated proteins podocin, nephrin, and synaptopodin and to enhanced transient receptor potential canonical 6 (TRPC6) channel expression and ATP-induced calcium influx in podocytes of Podo-GC-A KO mice. Concomitant treatment of Podo-GC-A KO mice with the TRPC channel blocker SKF96365 markedly ameliorated albuminuria and glomerular damage in response to DOCA. In conclusion, the physiologic effects of NPs on GFR and natriuresis do not involve podocytes. However, NP/GC-A/cGMP signaling protects podocyte integrity under pathologic conditions, most likely by suppression of TRPC channels. PMID:27153922

  18. Discovery of Potent Human Glutaminyl Cyclase Inhibitors as Anti-Alzheimer's Agents Based on Rational Design.

    PubMed

    Hoang, Van-Hai; Tran, Phuong-Thao; Cui, Minghua; Ngo, Van T H; Ann, Jihyae; Park, Jongmi; Lee, Jiyoun; Choi, Kwanghyun; Cho, Hanyang; Kim, Hee; Ha, Hee-Jin; Hong, Hyun-Seok; Choi, Sun; Kim, Young-Ho; Lee, Jeewoo

    2017-03-23

    Glutaminyl cyclase (QC) has been implicated in the formation of toxic amyloid plaques by generating the N-terminal pyroglutamate of β-amyloid peptides (pGlu-Aβ) and thus may participate in the pathogenesis of Alzheimer's disease (AD). We designed a library of glutamyl cyclase (QC) inhibitors based on the proposed binding mode of the preferred substrate, Aβ 3E-42 . An in vitro structure-activity relationship study identified several excellent QC inhibitors demonstrating 5- to 40-fold increases in potency compared to a known QC inhibitor. When tested in mouse models of AD, compound 212 significantly reduced the brain concentrations of pyroform Aβ and total Aβ and restored cognitive functions. This potent Aβ-lowering effect was achieved by incorporating an additional binding region into our previously established pharmacophoric model, resulting in strong interactions with the carboxylate group of Glu327 in the QC binding site. Our study offers useful insights in designing novel QC inhibitors as a potential treatment option for AD.

  19. Molecular determinants of Guanylate Cyclase Activating Protein subcellular distribution in photoreceptor cells of the retina.

    PubMed

    López-Begines, Santiago; Plana-Bonamaisó, Anna; Méndez, Ana

    2018-02-13

    Retinal guanylate cyclase (RetGC) and guanylate cyclase activating proteins (GCAPs) play an important role during the light response in photoreceptor cells. Mutations in these proteins are linked to distinct forms of blindness. RetGC and GCAPs exert their role at the ciliary outer segment where phototransduction takes place. We investigated the mechanisms governing GCAP1 and GCAP2 distribution to rod outer segments by expressing selected GCAP1 and GCAP2 mutants as transient transgenes in the rods of GCAP1/2 double knockout mice. We show that precluding GCAP1 direct binding to RetGC (K23D/GCAP1) prevented its distribution to rod outer segments, while preventing GCAP1 activation of RetGC post-binding (W94A/GCAP1) did not. We infer that GCAP1 translocation to the outer segment strongly depends on GCAP1 binding affinity for RetGC, which points to GCAP1 requirement to bind to RetGC to be transported. We gain further insight into the distinctive regulatory steps of GCAP2 distribution, by showing that a phosphomimic at position 201 is sufficient to retain GCAP2 at proximal compartments; and that the bovine equivalent to blindness-causative mutation G157R/GCAP2 results in enhanced phosphorylation in vitro and significant retention at the inner segment in vivo, as likely contributing factors to the pathophysiology.

  20. Binding of (/sup 3/H)forskolin to solubilized preparations of adenylate cyclase

    SciTech Connect

    Nelson, C.A.; Seamon, K.B.

    1988-01-01

    The binding of (/sup 3/H)forskolin to proteins solubilized from bovine brain membranes was studied by precipitating proteins with polyethylene glycol and separating (/sup 3/H)forskolin bound to protein from free (/sup 3/H)forskolin by rapid filtration. The K/sub d/ for (/sup 3/H)forskolin binding to solubilized proteins was 14 nM which was similar to that for (/sup 3/H)forskolin binding sites in membranes from rat brain and human platelets. Forskolin analogs competed for (/sup 3/H)forskolin binding sites with the same rank potency in both brain membranes and in proteins solubilized from brain membranes. (/sup 3/H)forskolin bound to proteins solubilized from membranes with a Bmaxmore » of 38 fmolmg protein which increased to 94 fmolmg protein when GppNHp was included in the binding assay. In contrast, GppNHp had no effect on (/sup 3/H)forskolin binding to proteins solubilized from membranes preactivated with GppNHp. Solubilized adenylate cyclase from non-preactivated membranes had a basal activity of 130 pmolmgmin which was increased 7-fold by GppNHp. In contrast, adenylate cyclase from preactivated membranes had a basal activity of 850 pmolmgmin which was not stimulated by GppNHp or forskolin« less

  1. A single gene for lycopene cyclase, phytoene synthase, and regulation of carotene biosynthesis in Phycomyces

    PubMed Central

    Arrach, Nabil; Fernández-Martín, Rafael; Cerdá-Olmedo, Enrique; Avalos, Javier

    2001-01-01

    Previous complementation and mapping of mutations that change the usual yellow color of the Zygomycete Phycomyces blakesleeanus to white or red led to the definition of two structural genes for carotene biosynthesis. We have cloned one of these genes, carRA, by taking advantage of its close linkage to the other, carB, responsible for phytoene dehydrogenase. The sequences of the wild type and six mutants have been established, compared with sequences in other organisms, and correlated with the mutant phenotypes. The carRA and carB coding sequences are separated by 1,381 untranslated nucleotides and are divergently transcribed. Gene carRA contains separate domains for two enzymes, lycopene cyclase and phytoene synthase, and regulates the overall activity of the pathway and its response to physical and chemical stimuli from the environment. The lycopene cyclase domain of carRA derived from a duplication of a gene from a common ancestor of fungi and Brevibacterium linens; the phytoene synthase domain is similar to the phytoene and squalene synthases of many organisms; but the regulatory functions appear to be specific to Phycomyces. PMID:11172012

  2. Association of Adenylate Cyclase 10 (ADCY10) Polymorphisms and Bone Mineral Density in Healthy Adults

    PubMed Central

    Ichikawa, Shoji; Koller, Daniel L.; Curry, Leah R.; Lai, Dongbing; Xuei, Xiaoling; Edenberg, Howard J.; Hui, Siu L.; Peacock, Munro; Foroud, Tatiana; Econs, Michael J.

    2010-01-01

    Phenotypic variation in bone mineral density (BMD) among healthy adults is influenced by both genetic and environmental factors. Genetic sequence variations in the adenylate cyclase 10 (ADCY10) gene, which is also called soluble adenylate cyclase, have previously been reported to be associated with low spinal BMD in hypercalciuric patients. Since ADCY10 is located in the region linked to spinal BMD in our previous linkage analysis, we tested whether polymorphisms in this gene are also associated with normal BMD variation in healthy adults. Sixteen single nucleotide polymorphisms (SNPs) distributed throughout ADCY10 were genotyped in two healthy groups of American whites: 1,692 premenopausal women and 715 men. Statistical analyses were performed in the two groups to test for association between these SNPs and femoral neck and lumbar spine areal BMD. We observed significant evidence of association (p<0.01) with one SNP each in men and women. Genotypes at these SNPs accounted for less than 1% of hip BMD variation in men, but 1.5% of spinal BMD in women. However, adjacent SNPs did not corroborate the association in either males or females. In conclusion, we found a modest association between an ADCY10 polymorphism and spinal areal BMD in premenopausal white women. PMID:19093065

  3. Hypoxic Vasospasm Mediated by cIMP: When Soluble Guanylyl Cyclase Turns Bad.

    PubMed

    Gao, Yuansheng; Chen, Zhengju; Leung, Susan W S; Vanhoutte, Paul M

    2015-06-01

    In a number of isolated blood vessel types, hypoxia causes an acute contraction that is dependent on the presence of nitric oxide and activation of soluble guanylyl cyclase. It is more pronounced when the preparations are constricted and is therefore termed hypoxic augmentation of vasoconstriction. This hypoxic response is accompanied by increases in the intracellular level of inosine 5'-triphosphate and in the synthesis of inosine 3',5'-cyclic monophosphate (cIMP) by soluble guanylyl cyclase. The administration of exogenous cIMP or inosine 5'-triphosphate causes augmented vasoconstriction to hypoxia. Furthermore, the vasoconstriction evoked by hypoxia and cIMP is associated with increased activity of Rho kinase (ROCK), indicating that cIMP may mediate the hypoxic effect by sensitizing the myofilaments to Ca through ROCK. Hypoxia is implicated in exaggerated vasoconstriction in the pathogenesis of coronary artery disease, myocardial infarction, hypertension, and stroke. The newly found role of cIMP may help to identify unique therapeutic targets for certain cardiovascular disorders.

  4. The Bordetella Adenylate Cyclase Repeat-in-Toxin (RTX) Domain Is Immunodominant and Elicits Neutralizing Antibodies*

    PubMed Central

    Wang, Xianzhe; Maynard, Jennifer A.

    2015-01-01

    The adenylate cyclase toxin (ACT) is a multifunctional virulence factor secreted by Bordetella species. Upon interaction of its C-terminal hemolysin moiety with the cell surface receptor αMβ2 integrin, the N-terminal cyclase domain translocates into the host cell cytosol where it rapidly generates supraphysiological cAMP concentrations, which inhibit host cell anti-bacterial activities. Although ACT has been shown to induce protective immunity in mice, it is not included in any current acellular pertussis vaccines due to protein stability issues and a poor understanding of its role as a protective antigen. Here, we aimed to determine whether any single domain could recapitulate the antibody responses induced by the holo-toxin and to characterize the dominant neutralizing antibody response. We first immunized mice with ACT and screened antibody phage display libraries for binding to purified ACT. The vast majority of unique antibodies identified bound the C-terminal repeat-in-toxin (RTX) domain. Representative antibodies binding two nonoverlapping, neutralizing epitopes in the RTX domain prevented ACT association with J774A.1 macrophages and soluble αMβ2 integrin, suggesting that these antibodies inhibit the ACT-receptor interaction. Sera from mice immunized with the RTX domain showed similar neutralizing activity as ACT-immunized mice, indicating that this domain induced an antibody response similar to that induced by ACT. These data demonstrate that RTX can elicit neutralizing antibodies and suggest it may present an alternative to ACT. PMID:25505186

  5. Expression, purification and crystallization of a plant polyketide cyclase from Cannabis sativa

    PubMed Central

    Yang, Xinmei; Matsui, Takashi; Mori, Takahiro; Taura, Futoshi; Noguchi, Hiroshi; Abe, Ikuro; Morita, Hiroyuki

    2015-01-01

    Plant polyketides are a structurally diverse family of natural products. In the biosynthesis of plant polyketides, the construction of the carbocyclic scaffold is a key step in diversifying the polyketide structure. Olivetolic acid cyclase (OAC) from Cannabis sativa L. is the only known plant polyketide cyclase that catalyzes the C2–C7 intramolecular aldol cyclization of linear pentyl tetra-β-ketide-CoA to generate olivetolic acid in the biosynthesis of cannabinoids. The enzyme is also thought to belong to the dimeric α+β barrel (DABB) protein family. However, because of a lack of functional analysis of other plant DABB proteins and low sequence identity with the functionally distinct bacterial DABB proteins, the catalytic mechanism of OAC has remained unclear. To clarify the intimate catalytic mechanism of OAC, the enzyme was overexpressed in Escherichia coli and crystallized using the vapour-diffusion method. The crystals diffracted X-rays to 1.40 Å resolution and belonged to space group P3121 or P3221, with unit-cell parameters a = b = 47.3, c = 176.0 Å. Further crystallographic analysis will provide valuable insights into the structure–function relationship and catalytic mechanism of OAC. PMID:26625288

  6. Expression, purification and crystallization of a plant polyketide cyclase from Cannabis sativa.

    PubMed

    Yang, Xinmei; Matsui, Takashi; Mori, Takahiro; Taura, Futoshi; Noguchi, Hiroshi; Abe, Ikuro; Morita, Hiroyuki

    2015-12-01

    Plant polyketides are a structurally diverse family of natural products. In the biosynthesis of plant polyketides, the construction of the carbocyclic scaffold is a key step in diversifying the polyketide structure. Olivetolic acid cyclase (OAC) from Cannabis sativa L. is the only known plant polyketide cyclase that catalyzes the C2-C7 intramolecular aldol cyclization of linear pentyl tetra-β-ketide-CoA to generate olivetolic acid in the biosynthesis of cannabinoids. The enzyme is also thought to belong to the dimeric α+β barrel (DABB) protein family. However, because of a lack of functional analysis of other plant DABB proteins and low sequence identity with the functionally distinct bacterial DABB proteins, the catalytic mechanism of OAC has remained unclear. To clarify the intimate catalytic mechanism of OAC, the enzyme was overexpressed in Escherichia coli and crystallized using the vapour-diffusion method. The crystals diffracted X-rays to 1.40 Å resolution and belonged to space group P3121 or P3221, with unit-cell parameters a = b = 47.3, c = 176.0 Å. Further crystallographic analysis will provide valuable insights into the structure-function relationship and catalytic mechanism of OAC.

  7. Oxygen sensation and social feeding mediated by a C. elegans guanylate cyclase homologue.

    PubMed

    Gray, Jesse M; Karow, David S; Lu, Hang; Chang, Andy J; Chang, Jennifer S; Ellis, Ronald E; Marletta, Michael A; Bargmann, Cornelia I

    2004-07-15

    Specialized oxygen-sensing cells in the nervous system generate rapid behavioural responses to oxygen. We show here that the nematode Caenorhabditis elegans exhibits a strong behavioural preference for 5-12% oxygen, avoiding higher and lower oxygen levels. 3',5'-cyclic guanosine monophosphate (cGMP) is a common second messenger in sensory transduction and is implicated in oxygen sensation. Avoidance of high oxygen levels by C. elegans requires the sensory cGMP-gated channel tax-2/tax-4 and a specific soluble guanylate cyclase homologue, gcy-35. The GCY-35 haem domain binds molecular oxygen, unlike the haem domains of classical nitric-oxide-regulated guanylate cyclases. GCY-35 and TAX-4 mediate oxygen sensation in four sensory neurons that control a naturally polymorphic social feeding behaviour in C. elegans. Social feeding and related behaviours occur only when oxygen exceeds C. elegans' preferred level, and require gcy-35 activity. Our results suggest that GCY-35 is regulated by molecular oxygen, and that social feeding can be a behavioural strategy for responding to hyperoxic environments.

  8. Understanding the Mechanism of Translocation of Adenylate Cyclase Toxin across Biological Membranes

    PubMed Central

    Ostolaza, Helena; Martín, César; González-Bullón, David; Uribe, Kepa B.; Etxaniz, Asier

    2017-01-01

    Adenylate cyclase toxin (ACT) is one of the principal virulence factors secreted by the whooping cough causative bacterium Bordetella pertussis, and it has a critical role in colonization of the respiratory tract and establishment of the disease. ACT targets phagocytes via binding to the CD11b/CD18 integrin and delivers its N-terminal adenylate cyclase (AC) domain directly to the cell cytosol, where it catalyzes unregulated conversion of cytosolic ATP into cAMP upon activation by binding to cellular calmodulin. High cAMP levels disrupt bactericidal functions of the immune cells, ultimately leading to cell death. In spite of its relevance in the ACT biology, the mechanism by which its ≈400 amino acid-long AC domain is transported through the target plasma membrane, and is released into the target cytosol, remains enigmatic. This article is devoted to refresh our knowledge on the mechanism of AC translocation across biological membranes. Two models, the so-called “two-step model” and the recently-proposed “toroidal pore model”, will be considered. PMID:28934133

  9. Investigation of cAMP microdomains as a path to novel cancer diagnostics.

    PubMed

    Desman, Garrett; Waintraub, Caren; Zippin, Jonathan H

    2014-12-01

    Understanding of cAMP signaling has greatly improved over the past decade. The advent of live cell imaging techniques and more specific pharmacologic modulators has led to an improved understanding of the intricacies by which cAMP is able to modulate such a wide variety of cellular pathways. It is now appreciated that cAMP is able to activate multiple effector proteins at distinct areas in the cell leading to the activation of very different downstream targets. The investigation of signaling proteins in cancer is a common route to the development of diagnostic tools, prognostic tools, and/or therapeutic targets, and in this review we highlight how investigation of cAMP signaling microdomains driven by the soluble adenylyl cyclase in different cancers has led to the development of a novel cancer biomarker. Antibodies directed against the soluble adenylyl cyclase (sAC) are highly specific markers for melanoma especially for lentigo maligna melanoma and are being described as "second generation" cancer diagnostics, which are diagnostics that determine the 'state' of a cell and not just identify the cell type. Due to the wide presence of cAMP signaling pathways in cancer, we predict that further investigation of both sAC and other cAMP microdomains will lead to additional cancer biomarkers. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. cAMP and Mitochondria

    PubMed Central

    Valsecchi, Federica; Ramos-Espiritu, Lavoisier S.; Buck, Jochen; Levin, Lonny R.

    2013-01-01

    Phosphorylation of mitochondrial proteins has emerged as a major regulatory mechanism for metabolic adaptation. cAMP signaling and PKA phosphorylation of mitochondrial proteins have just started to be investigated, and the presence of cAMP-generating enzymes and PKA inside mitochondria is still controversial. Here, we discuss the role of cAMP in regulating mitochondrial bioenergetics through protein phosphorylation and the evidence for soluble adenylyl cyclase as the source of cAMP inside mitochondria. PMID:23636265

  11. (S)Pot on Mitochondria: Cannabinoids Disrupt Cellular Respiration to Limit Neuronal Activity.

    PubMed

    Harkany, Tibor; Horvath, Tamas L

    2017-01-10

    Classical views posit G protein-coupled cannabinoid receptor 1s (CB1Rs) at the cell surface with cytosolic Giα-mediated signal transduction. Hebert-Chatelain et al. (2016) instead place CB 1 Rs at mitochondria limiting neuronal respiration by soluble adenylyl cyclase-dependent modulation of complex I activity. Thus, neuronal bioenergetics link to synaptic plasticity and, globally, learning and memory. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp.

    PubMed

    Gao, Shiqiang; Nagpal, Jatin; Schneider, Martin W; Kozjak-Pavlovic, Vera; Nagel, Georg; Gottschalk, Alexander

    2015-09-08

    Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s(-1)). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals.

  13. Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp

    PubMed Central

    Gao, Shiqiang; Nagpal, Jatin; Schneider, Martin W.; Kozjak-Pavlovic, Vera; Nagel, Georg; Gottschalk, Alexander

    2015-01-01

    Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s−1). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals. PMID:26345128

  14. On the role of adenylate cyclase, tyrosine kinase, and tyrosine phosphatase in the response of nerve and glial cells to photodynamic impact

    NASA Astrophysics Data System (ADS)

    Kolosov, Mikhail S.; Bragin, D. E.; Dergacheva, Olga Y.; Vanzha, O.; Oparina, L.; Uzdensky, Anatoly B.

    2004-08-01

    The role of different intercellular signaling pathways involving adenylate cyclase (AC), receptor tyrosine kinase (RTK), tyrosine and serine/threonine protein phosphatases (PTP or PP, respectively) in the response of crayfish mechanoreceptor neuron (MRN) and surrounding glial cells to photodynamic effect of aluminum phthalocyanine Photosens have been studied. AC inhibition by MDL-12330A decreased neuron lifetime, whereas AC activation by forskolin increase it. Thus, increase in cAMP produced by activated AC protects SRN against photodynamic inactivation. Similarly, RTK inhibition by genistein decreased neuron lifetime, while inhibition of PTP or PP that remove phosphate groups from proteins, prolonged neuronal activity. AC inhibition reduced photoinduced damage of the plasma membrane, and, therefore, necrosis in neuronal and glial cells. RTK inhibition protected only neurons against PDT-induced membrane permeabilization while glial cells became lesser permeable under ortovanadate-mediated PTP inhibition. AC activation also prevented PDT-induced apoptosis in glial cells. PP inhibition enhanced apoptotic processes in photosensitized glial cells. Therefore, both intercellular signaling pathways involving AC and TRK are involved in the maintenance of neuronal activity, integrity of the neuronal and glial plasma membranes and in apoptotic processes in glia under photosensitization.

  15. Necrotic enteritis locus 1 diguanylate cyclase and phosphodiesterase (cyclic-di-GMP) gene mutation attenuates virulence in an avian necrotic enteritis isolate of Clostridium perfringens.

    PubMed

    Parreira, Valeria R; Ojha, Shivani; Lepp, Dion; Mehdizadeh Gohari, Iman; Zhou, Hongzhuan; Susta, Leonardo; Gong, Jianhua; Prescott, John F

    2017-09-01

    Necrotic enteritis (NE) caused by netB-positive strains of Clostridium perfringens is an important disease of intensively-reared broiler chickens. It is widely controlled by antibiotic use, but this practice that has come under increasing scrutiny and alternative approaches are required. As part of the search for alternative approaches over the last decade, advances have been made in understanding its pathogenesis but much remains to be understood and applied to the control of NE. The objective of this work was to assess the effect on virulence of mutation of the cyclic-di-GMP signaling genes present on the large pathogenicity locus (NELoc-1) in the tcp-encoding conjugative virulence plasmid, pNetB. For this purpose, the diguanylate cyclase (dgc) and phosphodiesterase (pde) genes were individually insertionally inactivated and the two mutants were subsequently complemented with their respective genes. Southern blotting showed that a single gene insertion was present. Mutation of either gene resulted in almost total attenuation of the mutants to cause NE in experimentally-infected broiler chickens, which was fully restored in each case by complementation of the respective mutated gene. Production of NetB-associated cytotoxicity for Leghorn male hepatoma (LMH) cells was unaffected in mutants. We conclude that the cyclic-di-GMP signaling system is important in controlling virulence in a NE C. perfringens strain and might be a target for control of the disease. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. A Single Oxidosqualene Cyclase Produces the Seco-Triterpenoid α-Onocerin1[OPEN

    PubMed Central

    Almeida, Aldo; Khakimov, Bekzod; Bassard, Jean-Etienne; Appendino, Giovanni

    2018-01-01

    8,14-seco-Triterpenoids are characterized by their unusual open C-ring. Their distribution in nature is rare and scattered in taxonomically unrelated plants. The 8,14-seco-triterpenoid α-onocerin is only known from the evolutionarily distant clubmoss genus Lycopodium and the leguminous genus Ononis, which makes the biosynthesis of this seco-triterpenoid intriguing from an evolutionary standpoint. In our experiments with Ononis spinosa, α-onocerin was detected only in the roots. Through transcriptome analysis of the roots, an oxidosqualene cyclase, OsONS1, was identified that produces α-onocerin from squalene-2,3;22,23-dioxide when transiently expressed in Nicotiana bethamiana. In contrast, in Lycopodium clavatum, two sequential cyclases, LcLCC and LcLCD, are required to produce α-onocerin in the N. benthamiana transient expression system. Expression of OsONS1 in the lanosterol synthase knockout yeast strain GIL77, which accumulates squalene-2,3;22,23-dioxide, verified the α-onocerin production. A phylogenetic analysis predicts that OsONS1 branches off from specific lupeol synthases and does not group with the known L. clavatum α-onocerin cyclases. Both the biochemical and phylogenetic analyses of OsONS1 suggest convergent evolution of the α-onocerin pathways. When OsONS1 was coexpressed in N. benthamiana leaves with either of the two O. spinosa squalene epoxidases, OsSQE1 or OsSQE2, α-onocerin production was boosted, most likely because the epoxidases produce higher amounts of squalene-2,3;22,23-dioxide. Fluorescence lifetime imaging microscopy analysis demonstrated specific protein-protein interactions between OsONS1 and both O. spinosa squalene epoxidases. Coexpression of OsONS1 with the two OsSQEs suggests that OsSQE2 is the preferred partner of OsONS1 in planta. Our results provide an example of the convergent evolution of plant specialized metabolism. PMID:29203557

  17. Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes.

    PubMed

    Wu, Vincent W; Dana, Craig M; Iavarone, Anthony T; Clark, Douglas S; Glass, N Louise

    2017-01-17

    The breakdown of plant biomass to simple sugars is essential for the production of second-generation biofuels and high-value bioproducts. Currently, enzymes produced from filamentous fungi are used for deconstructing plant cell wall polysaccharides into fermentable sugars for biorefinery applications. A post-translational N-terminal pyroglutamate modification observed in some of these enzymes occurs when N-terminal glutamine or glutamate is cyclized to form a five-membered ring. This modification has been shown to confer resistance to thermal denaturation for CBH-1 and EG-1 cellulases. In mammalian cells, the formation of pyroglutamate is catalyzed by glutaminyl cyclases. Using the model filamentous fungus Neurospora crassa, we identified two genes (qc-1 and qc-2) that encode proteins homologous to mammalian glutaminyl cyclases. We show that qc-1 and qc-2 are essential for catalyzing the formation of an N-terminal pyroglutamate on CBH-1 and GH5-1. CBH-1 and GH5-1 produced in a Δqc-1 Δqc-2 mutant, and thus lacking the N-terminal pyroglutamate modification, showed greater sensitivity to thermal denaturation, and for GH5-1, susceptibility to proteolytic cleavage. QC-1 and QC-2 are endoplasmic reticulum (ER)-localized proteins. The pyroglutamate modification is predicted to occur in a number of additional fungal proteins that have diverse functions. The identification of glutaminyl cyclases in fungi may have implications for production of lignocellulolytic enzymes, heterologous expression, and biotechnological applications revolving around protein stability. Pyroglutamate modification is the post-translational conversion of N-terminal glutamine or glutamate into a cyclized amino acid derivative. This modification is well studied in animal systems but poorly explored in fungal systems. In Neurospora crassa, we show that this modification takes place in the ER and is catalyzed by two well-conserved enzymes, ubiquitously conserved throughout the fungal kingdom. We

  18. Characterization of the homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase.

    PubMed

    Dix, C J; Habberfield, A D; Cooke, B A

    1984-06-15

    The homologous and heterologous desensitization of rat Leydig-tumour-cell adenylate cyclase induced by lutropin (LH) was characterized with the aid of forskolin and cholera toxin. Forskolin stimulated cyclic AMP production in a dose-dependent manner, with linear kinetics up to 2h. Forskolin also potentiated the action of LH on cyclic AMP production, but was only additive with cholera toxin. Preincubation of rat Leydig tumour cells with LH (1.0 micrograms/ml) for 1 h produced a desensitization of the subsequent LH (1.0 micrograms/ml)-stimulated cyclic AMP production, whereas the responses to cholera toxin (5.0 micrograms/ml), forskolin (100 microM), LH plus forskolin or cholera toxin plus forskolin were unaltered. In contrast, preincubation with LH for 20h produced a desensitization to all the stimuli tested. When rat Leydig tumour cells were preincubated for 1h with forskolin or dibutyryl cyclic AMP, the only subsequent response that was significantly altered was that to LH plus forskolin after preincubation with forskolin. However, preincubation for 20h with forskolin or dibutyryl cyclic AMP induced a desensitization to all stimuli subsequently tested. LH produced a rapid (0-1h) homologous desensitization, which was followed by a slower (2-8h)-onset heterologous desensitization. Forskolin and dibutyryl cyclic AMP were only able to induce heterologous desensitization. The rate of desensitization induced by either forskolin or dibutyryl cyclic AMP was similar to the rate of heterologous desensitization induced by LH. These results demonstrate that in purified rat Leydig tumour cells LH produces an initial homologous desensitization of adenylate cyclase that involves a cyclic AMP-independent lesion at or proximal to the guanine nucleotide regulatory protein (G-protein). This is followed by heterologous desensitization, which can also be induced by forskolin or dibutyryl cyclic AMP, thus indicating that LH-induced heterologous desensitization of rat Leydig

  19. Characterization of plant carotenoid cyclases as members of the flavoprotein family functioning with no net redox change.

    PubMed

    Mialoundama, Alexis Samba; Heintz, Dimitri; Jadid, Nurul; Nkeng, Paul; Rahier, Alain; Deli, Jozsef; Camara, Bilal; Bouvier, Florence

    2010-07-01

    The later steps of carotenoid biosynthesis involve the formation of cyclic carotenoids. The reaction is catalyzed by lycopene beta-cyclase (LCY-B), which converts lycopene into beta-carotene, and by capsanthin-capsorubin synthase (CCS), which is mainly dedicated to the synthesis of kappa-cyclic carotenoids (capsanthin and capsorubin) but also has LCY-B activity. Although the peptide sequences of plant LCY-Bs and CCS contain a putative dinucleotide-binding motif, it is believed that these two carotenoid cyclases proceed via protic activation and stabilization of resulting carbocation intermediates. Using pepper (Capsicum annuum) CCS as a prototypic carotenoid cyclase, we show that the monomeric protein contains one noncovalently bound flavin adenine dinucleotide (FAD) that is essential for enzyme activity only in the presence of NADPH, which functions as the FAD reductant. The reaction proceeds without transfer of hydrogen from the dinucleotide cofactors to beta-carotene or capsanthin. Using site-directed mutagenesis, amino acids potentially involved in the protic activation were identified. Substitutions of alanine, lysine, and arginine for glutamate-295 in the conserved 293-FLEET-297 motif of pepper CCS or LCY-B abolish the formation of beta-carotene and kappa-cyclic carotenoids. We also found that mutations of the equivalent glutamate-196 located in the 194-LIEDT-198 domain of structurally divergent bacterial LCY-B abolish the formation of beta-carotene. The data herein reveal plant carotenoid cyclases to be novel enzymes that combine characteristics of non-metal-assisted terpene cyclases with those attributes typically found in flavoenzymes that catalyze reactions, with no net redox, such as type 2 isopentenyl diphosphate isomerase. Thus, FAD in its reduced form could be implicated in the stabilization of the carbocation intermediate.

  20. Involvement of endogenous antioxidant systems in the protective activity of pituitary adenylate cyclase-activating polypeptide against hydrogen peroxide-induced oxidative damages in cultured rat astrocytes.

    PubMed

    Douiri, Salma; Bahdoudi, Seyma; Hamdi, Yosra; Cubì, Roger; Basille, Magali; Fournier, Alain; Vaudry, Hubert; Tonon, Marie-Christine; Amri, Mohamed; Vaudry, David; Masmoudi-Kouki, Olfa

    2016-06-01

    Astroglial cells possess an array of cellular defense mechanisms, including superoxide dismutase (SOD) and catalase antioxidant enzymes, to prevent damages caused by oxidative stress. Nevertheless, astroglial cell viability and functionality can be affected by significant oxidative stress. We have previously shown that pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent glioprotective agent that prevents hydrogen peroxide (H2 O2 )-induced apoptosis in cultured astrocytes. The purpose of this study was to investigate the potential protective effect of PACAP against oxidative-generated alteration of astrocytic antioxidant systems. Incubation of cells with subnanomolar concentrations of PACAP inhibited H2 O2 -evoked reactive oxygen species accumulation, mitochondrial respiratory burst, and caspase-3 mRNA level increase. PACAP also stimulated SOD and catalase activities in a concentration-dependent manner, and counteracted the inhibitory effect of H2 O2 on the activity of these two antioxidant enzymes. The protective action of PACAP against H2 O2 -evoked inhibition of antioxidant systems in astrocytes was protein kinase A, PKC, and MAP-kinase dependent. In the presence of H2 O2 , the SOD blocker NaCN and the catalase inhibitor 3-aminotriazole, both suppressed the protective effects of PACAP on SOD and catalase activities, mitochondrial function, and cell survival. Taken together, these results indicate that the anti-apoptotic effect of PACAP on astroglial cells can account for the activation of endogenous antioxidant enzymes and reduction in respiration rate, thus preserving mitochondrial integrity and preventing caspase-3 expression provoked by oxidative stress. Considering its powerful anti-apoptotic and anti-oxidative properties, the PACAPergic signaling system should thus be considered for the development of new therapeutical approaches to cure various pathologies involving oxidative neurodegeneration. We propose the following cascade for the

  1. Reconstitution of a fungal meroterpenoid biosynthesis reveals the involvement of a novel family of terpene cyclases

    NASA Astrophysics Data System (ADS)

    Itoh, Takayuki; Tokunaga, Kinya; Matsuda, Yudai; Fujii, Isao; Abe, Ikuro; Ebizuka, Yutaka; Kushiro, Tetsuo

    2010-10-01

    Meroterpenoids are hybrid natural products of both terpenoid and polyketide origin. We identified a biosynthetic gene cluster that is responsible for the production of the meroterpenoid pyripyropene in the fungus Aspergillus fumigatus through reconstituted biosynthesis of up to five steps in a heterologous fungal expression system. The cluster revealed a previously unknown terpene cyclase with an unusual sequence and protein primary structure. The wide occurrence of this sequence in other meroterpenoid and indole-diterpene biosynthetic gene clusters indicates the involvement of these enzymes in the biosynthesis of various terpenoid-bearing metabolites produced by fungi and bacteria. In addition, a novel polyketide synthase that incorporated nicotinyl-CoA as the starter unit and a prenyltransferase, similar to that in ubiquinone biosynthesis, was found to be involved in the pyripyropene biosynthesis. The successful production of a pyripyropene analogue illustrates the catalytic versatility of these enzymes for the production of novel analogues with useful biological activities.

  2. Structure-activity relationships of benzimidazole-based glutaminyl cyclase inhibitors featuring a heteroaryl scaffold.

    PubMed

    Ramsbeck, Daniel; Buchholz, Mirko; Koch, Birgit; Böhme, Livia; Hoffmann, Torsten; Demuth, Hans-Ulrich; Heiser, Ulrich

    2013-09-12

    Glutaminyl cyclase (hQC) has emerged as a new potential target for the treatment of Alzheimer's disease (AD). The inhibition of hQC prevents of the formation of the Aβ3(pE)-40,42 species which were shown to be of elevated neurotoxicity and are likely to act as a seeding core, leading to an accelerated formation of Aβ-oligomers and fibrils. This work presents a new class of inhibitors of hQC, resulting from a pharmacophore-based screen. Hit molecules were identified, containing benzimidazole as the metal binding group connected to 1,3,4-oxadiazole as the central scaffold. The subsequent optimization resulted in benzimidazolyl-1,3,4-thiadiazoles and -1,2,3-triazoles with an inhibitory potency in the nanomolar range. Further investigation into the potential binding mode of the new compound classes combined molecular docking and site directed mutagenesis studies.

  3. Lycopene cyclase paralog CruP protects against reactive oxygen species in oxygenic photosynthetic organisms.

    PubMed

    Bradbury, Louis M T; Shumskaya, Maria; Tzfadia, Oren; Wu, Shi-Biao; Kennelly, Edward J; Wurtzel, Eleanore T

    2012-07-03

    In photosynthetic organisms, carotenoids serve essential roles in photosynthesis and photoprotection. A previous report designated CruP as a secondary lycopene cyclase involved in carotenoid biosynthesis [Maresca J, et al. (2007) Proc Natl Acad Sci USA 104:11784-11789]. However, we found that cruP KO or cruP overexpression plants do not exhibit correspondingly reduced or increased production of cyclized carotenoids, which would be expected if CruP was a lycopene cyclase. Instead, we show that CruP aids in preventing accumulation of reactive oxygen species (ROS), thereby reducing accumulation of β-carotene-5,6-epoxide, a ROS-catalyzed autoxidation product, and inhibiting accumulation of anthocyanins, which are known chemical indicators of ROS. Plants with a nonfunctional cruP accumulate substantially higher levels of ROS and β-carotene-5,6-epoxide in green tissues. Plants overexpressing cruP show reduced levels of ROS, β-carotene-5,6-epoxide, and anthocyanins. The observed up-regulation of cruP transcripts under photoinhibitory and lipid peroxidation-inducing conditions, such as high light stress, cold stress, anoxia, and low levels of CO(2), fits with a role for CruP in mitigating the effects of ROS. Phylogenetic distribution of CruP in prokaryotes showed that the gene is only present in cyanobacteria that live in habitats characterized by large variation in temperature and inorganic carbon availability. Therefore, CruP represents a unique target for developing resilient plants and algae needed to supply food and biofuels in the face of global climate change.

  4. Tachyphylaxis to PACAP-27 after inhibition of NO synthesis: a loss of adenylate cyclase activation

    NASA Technical Reports Server (NTRS)

    Whalen, E. J.; Johnson, A. K.; Lewis, S. J.

    1999-01-01

    The vasodilator effects of pituitary adenylate cyclase activating polypeptide (PACAP-27) are subject to tachyphylaxis in rats treated with the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME). This study examined whether this tachyphylaxis is due to the loss of vasodilator potency of cAMP generated by activation of the G(s) protein-coupled PACAP receptors. Five successive treatments with PACAP-27 (2 nmol/kg iv) produced pronounced vasodilator responses in saline-treated rats that were not subject to tachyphylaxis. The first injection of PACAP-27 (2 nmol/kg iv) in L-NAME (50 micromol/kg iv)-treated rats produced vasodilator responses of similar magnitude to those in saline-treated rats, whereas four subsequent injections produced progressively and markedly smaller responses. The hemodynamic effects of the membrane-permeable cAMP analog 8-(4-chlorophenylthiol)-cAMP (8-CPT-cAMP; 5-15 micromol/kg iv) were similar in L-NAME-treated rats and in L-NAME-treated rats that had received the five injections of PACAP-27. In addition, five injections of 8-CPT-cAMP (10 micromol/kg iv) produced pronounced vasodilator responses in saline- and L-NAME-treated rats that were not subject to the development of tachyphylaxis. These results suggest that a loss of biological potency of cAMP is not responsible for tachyphylaxis to PACAP-27 in L-NAME-treated rats. This tachyphylaxis may be due to the inability of the G(s) protein-coupled PACAP receptor to activate adenylate cyclase.

  5. Docosahexaenoic acid alters Gsα localization in lipid raft and potentiates adenylate cyclase.

    PubMed

    Zhu, Zhuoran; Tan, Zhoubin; Li, Yan; Luo, Hongyan; Hu, Xinwu; Tang, Ming; Hescheler, Jürgen; Mu, Yangling; Zhang, Lanqiu

    2015-01-01

    Supplementation with docosahexaenoic acid (DHA), an ω-3 polyunsaturated fatty acid (PUFA), recently has become popular for the amelioration of depression; however the molecular mechanism of DHA action remains unclear. The aim of this study was to investigate the mechanism underlying the antidepressant effect of DHA by evaluating Gsα localization in lipid raft and the activity of adenylate cyclase in an in vitro glioma cell model. Lipid raft fractions from C6 glioma cells treated chronically with DHA were isolated by sucrose gradient ultracentrifugation. The content of Gsα in lipid raft was analyzed by immunoblotting and colocalization of Gsα with lipid raft was subjected to confocal microscopic analysis. The intracellular cyclic adenosine monophosphate (cAMP) level was determined by cAMP immunoassay kit. DHA decreased the amount of Gsα in lipid raft, whereas whole cell lysate Gsα was not changed. Confocal microscopic analysis demonstrated that colocalization of Gsα with lipid raft was decreased, whereas DHA increased intracellular cAMP accumulation in a dose-dependent manner. Interestingly, we found that DHA increased the lipid raft level, instead of disrupting it. The results of this study suggest that DHA may exert its antidepressant effect by translocating Gsα from lipid raft and potentiating the activity of adenylate cyclase. Importantly, the reduced Gsα in lipid raft by DHA is independent of disruption of lipid raft. Overall, the study provides partial preclinical evidence supporting a safe and effective therapy using DHA for depression. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Aniracetam improves behavioural responses and facilitates signal transduction in the rat brain.

    PubMed

    Ventra, C; Grimaldi, M; Meucci, O; Scorziello, A; Apicella, A; Filetti, E; Marino, A; Schettini, G

    1994-01-01

    The effect of aniracetam (10, 50, 100 mg/kg i.p. daily for 15 days) on both behavioural and biochemical parameters was investigated in the adult rat. Animals given aniracetam (50 mg/kg 1 h before the trial) showed a significant increase in the percentage of conditioned active avoidance responses and a reduction of latency times. Aniracetam significantly counteracted the scopolamine-induced memory failure at the passive avoidance (step down) test, while it did not modify the locomotion of the animals. In purified frontocortical and hippocampal synaptic membranes of rats treated with aniracetam (50 mg/kg i.p. daily for 15 days) a potentiation of basal, carbamylcholine-, dopamine- and norepinephrine-stimulated adenylyl cyclase activity was observed, while forskolin-stimulated enzyme activity was not modified. With regard to inositol phosphate production measured in fronto-cortical synaptoneurosomes, aniracetam potentiated the stimulation by angiotensin II, while the stimulation by carbamylcholine, not affected by 10 and 50 mg/kg aniracetam, was notably, although not significantly, decreased by 100 mg/kg aniracetam. Furthermore, in synaptosomes derived from hippocampus, aniracetam (50 mg/kg i.p. daily for 15 days) caused an increase of both basal and K(+)-stimulated intrasynaptosomal Ca(2+) concentration. In conclusion, a correlation between the improvement of behavioural performance and the modulation of transducing systems by aniracetam seems to take place in brain areas, such as frontal cortex and hippocampus, known to play a major role in the control of cognitive functions.

  7. Hypercapnia modulates cAMP signalling and cystic fibrosis transmembrane conductance regulator‐dependent anion and fluid secretion in airway epithelia

    PubMed Central

    Turner, Mark J.; Saint‐Criq, Vinciane; Patel, Waseema; Ibrahim, Salam H.; Verdon, Bernard; Ward, Christopher; Garnett, James P.; Tarran, Robert; Cann, Martin J.

    2015-01-01

    Key points Raised arterial blood CO2 (hypercapnia) is a feature of many lung diseases.CO2 has been shown to act as a cell signalling molecule in human cells, notably by influencing the levels of cell signalling second messengers: cAMP and Ca2+.Hypercapnia reduced cAMP‐stimulated cystic fibrosis transmembrane conductance regulator‐dependent anion and fluid transport in Calu‐3 cells and primary human airway epithelia but did not affect cAMP‐regulated HCO3 − transport via pendrin or Na+/HCO3 − cotransporters.These results further support the role of CO2 as a cell signalling molecule and suggests CO2‐induced reductions in airway anion and fluid transport may impair innate defence mechanisms of the lungs. Abstract Hypercapnia is clinically defined as an arterial blood partial pressure of CO2 of above 40 mmHg and is a feature of chronic lung disease. In previous studies we have demonstrated that hypercapnia modulates agonist‐stimulated cAMP levels through effects on transmembrane adenylyl cyclase activity. In the airways, cAMP is known to regulate cystic fibrosis transmembrane conductance regulator (CFTR)‐mediated anion and fluid secretion, which contributes to airway surface liquid homeostasis. The aim of the current work was to investigate if hypercapnia could modulate cAMP‐regulated ion and fluid transport in human airway epithelial cells. We found that acute exposure to hypercapnia significantly reduced forskolin‐stimulated elevations in intracellular cAMP as well as both adenosine‐ and forskolin‐stimulated increases in CFTR‐dependent transepithelial short‐circuit current, in polarised cultures of Calu‐3 human airway cells. This CO2‐induced reduction in anion secretion was not due to a decrease in HCO3 − transport given that neither a change in CFTR‐dependent HCO3 − efflux nor Na+/HCO3 − cotransporter‐dependent HCO3 − influx were CO2‐sensitive. Hypercapnia also reduced the volume of forskolin‐stimulated fluid

  8. Period1 gates the circadian modulation of memory-relevant signaling in mouse hippocampus by regulating the nuclear shuttling of the CREB kinase pP90RSK.

    PubMed

    Rawashdeh, Oliver; Jilg, Antje; Maronde, Erik; Fahrenkrug, Jan; Stehle, Jörg H

    2016-09-01

    Memory performance varies over a 24-h day/night cycle. While the detailed underlying mechanisms are yet unknown, recent evidence suggests that in the mouse hippocampus, rhythmic phosphorylation of mitogen-activated protein kinase (MAPK) and cyclic adenosine monophosphate response element-binding protein (CREB) are central to the circadian (~ 24 h) regulation of learning and memory. We recently identified the clock protein PERIOD1 (PER1) as a vehicle that translates information encoding time of day to hippocampal plasticity. We here elaborate how PER1 may gate the sensitivity of memory-relevant hippocampal signaling pathways. We found that in wild-type mice (WT), spatial learning triggers CREB phosphorylation only during the daytime, and that this effect depends on the presence of PER1. The time-of-day-dependent induction of CREB phosphorylation can be reproduced pharmacologically in acute hippocampal slices prepared from WT mice, but is absent in preparations made from Per1-knockout (Per1(-/-) ) mice. We showed that the PER1-dependent CREB phosphorylation is regulated downstream of MAPK. Stimulation of WT hippocampal neurons triggered the co-translocation of PER1 and the CREB kinase pP90RSK (pMAPK-activated ribosomal S6 kinase) into the nucleus. In hippocampal neurons from Per1(-/-) mice, however, pP90RSK remained perinuclear. A co-immunoprecipitation assay confirmed a high-affinity interaction between PER1 and pP90RSK. Knocking down endogenous PER1 in hippocampal cells inhibited adenylyl cyclase-dependent CREB activation. Taken together, the PER1-dependent modulation of cytoplasmic-to-nuclear signaling in the murine hippocampus provides a molecular explanation for how the circadian system potentially shapes a temporal framework for daytime-dependent memory performance, and adds a novel facet to the versatility of the clock gene protein PER1. We provide evidence that the circadian clock gene Period1 (Per1) regulates CREB phosphorylation in the mouse hippocampus

  9. A Japanese family with nonautoimmune hyperthyroidism caused by a novel heterozygous thyrotropin receptor gene mutation.

    PubMed

    Nakamura, Akie; Morikawa, Shuntaro; Aoyagi, Hayato; Ishizu, Katsura; Tajima, Toshihiro

    2014-06-01

    Hyperthyroidism caused by activating mutations of the thyrotropin receptor gene (TSHR) is rare in the pediatric population. We found a Japanese family with hyperthyroidism without autoantibody. DNA sequence analysis of TSHR was undertaken in this family. The functional consequences for the Gs-adenylyl cyclase and Gq/11-phospholipase C signaling pathways and cell surface expression of receptors were determined in vitro using transiently transfected human embryonic kidney 293 cells. We identified a heterozygous mutation (M453R) in exon 10 of TSHR. In this family, this mutation was found in all individuals who exhibited hyperthyroidism. The results showed that this mutation resulted in constitutive activation of the Gs-adenylyl cyclase system. However, this mutation also caused a reduction in the activation capacity of the Gq/11-phospholipase C pathway, compared with the wild type. We demonstrate that the M453R mutation is the cause of nonautoimmune hyperthyroidism.

  10. The signaling helix: a common functional theme in diverse signaling proteins

    PubMed Central

    Anantharaman, Vivek; Balaji, S; Aravind, L

    2006-01-01

    Background The mechanism by which the signals are transmitted between receptor and effector domains in multi-domain signaling proteins is poorly understood. Results Using sensitive sequence analysis methods we identify a conserved helical segment of around 40 residues in a wide range of signaling proteins, including numerous sensor histidine kinases such as Sln1p, and receptor guanylyl cyclases such as the atrial natriuretic peptide receptor and nitric oxide receptors. We term this helical segment the signaling (S)-helix and present evidence that it forms a novel parallel coiled-coil element, distinct from previously known helical segments in signaling proteins, such as the Dimerization-Histidine phosphotransfer module of histidine kinases, the intra-cellular domains of the chemotaxis receptors, inter-GAF domain helical linkers and the α-helical HAMP module. Analysis of domain architectures allowed us to reconstruct the domain-neighborhood graph for the S-helix, which showed that the S-helix almost always occurs between two signaling domains. Several striking patterns in the domain neighborhood of the S-helix also became evident from the graph. It most often separates diverse N-terminal sensory domains from various C-terminal catalytic signaling domains such as histidine kinases, cNMP cyclase, PP2C phosphatases, NtrC-like AAA+ ATPases and diguanylate cyclases. It might also occur between two sensory domains such as PAS domains and occasionally between a DNA-binding HTH domain and a sensory domain. The sequence conservation pattern of the S-helix revealed the presence of a unique constellation of polar residues in the dimer-interface positions within the central heptad of the coiled-coil formed by the S-helix. Conclusion Combining these observations with previously reported mutagenesis studies on different S-helix-containing proteins we suggest that it functions as a switch that prevents constitutive activation of linked downstream signaling domains. However, upon

  11. Serotonin-Sensitive Adenylate Cyclase in Neural Tissue and Its Similarity to the Serotonin Receptor: A Possible Site of Action of Lysergic Acid Diethylamide

    PubMed Central

    Nathanson, James A.; Greengard, Paul

    1974-01-01

    An adenylate cyclase (EC 4.6.1.1) that is activated specifically by low concentrations of serotonin has been identified in homogenates of the thoracic ganglia of an insect nervous system. The activation of this enzyme by serotonin was selectively inhibited by extremely low concentrations of D-lysergic acid diethylamide (LSD), 2-bromo-LSD, and cyproheptadine, agents which are known to block certain serotonin receptors in vivo. The inhibition was competitive with respect to serotonin, and the calculated inhibitory constant of LSD for this serotonin-sensitive adenylate cyclase was 5 nM. The data are consistent with a model in which the serotonin receptor of neural tissue is intimately associated with a serotonin-sensitive adenylate cyclase which mediates serotonergic neurotransmission. The results are also compatible with the possibility that some of the physiological effects of LSD may be mediated through interaction with serotonin-sensitive adenylate cyclase. PMID:4595572

  12. Chronic changes in pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulation in rats.

    PubMed

    Han, Xun; Ran, Ye; Su, Min; Liu, Yinglu; Tang, Wenjing; Dong, Zhao; Yu, Shengyuan

    2017-01-01

    Background Preclinical experimental studies revealed an acute alteration of pituitary adenylate cyclase-activating polypeptide in response to a single activation of the trigeminovascular system, which suggests a potential role of pituitary adenylate cyclase-activating polypeptide in the pathogenesis of migraine. However, changes in pituitary adenylate cyclase-activating polypeptide after repeated migraine-like attacks in chronic migraine are not clear. Therefore, the present study investigated chronic changes in pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulations in the rat. Methods A rat model of chronic migraine was established by repeated chemical dural stimulations using an inflammatory soup for a different numbers of days. The pituitary adenylate cyclase-activating polypeptide levels were quantified in plasma, the trigeminal ganglia, and the trigeminal nucleus caudalis using radioimmunoassay and Western blotting in trigeminal ganglia and trigeminal nucleus caudalis tissues. Western blot analysis and real-time polymerase chain reaction were used to measure the protein and mRNA expression of pituitary adenylate cyclase-activating polypeptide-related receptors (PAC1, VPAC1, and VPAC2) in the trigeminal ganglia and trigeminal nucleus caudalis to identify changes associated with repetitive applications of chemical dural stimulations. Results All rats exhibited significantly decreased periorbital nociceptive thresholds to repeated inflammatory soup stimulations. Radioimmunoassay and Western blot analysis demonstrated significantly decreased pituitary adenylate cyclase-activating polypeptide levels in plasma and trigeminal ganglia after repetitive chronic inflammatory soup stimulation. Protein and mRNA analyses of pituitary adenylate cyclase-activating polypeptide-related receptors demonstrated significantly increased PAC1 receptor protein and mRNA expression in the trigeminal ganglia, but not

  13. Heavy isotope labeling study of the turnover of forskolin-stimulated adenylate cyclase in BC/sup 3/H1 cell line

    SciTech Connect

    Bouhelal, R.; Bockaert, J.; Mermet-Bouvier, R.

    1987-06-25

    We have used the method of heavy isotope labeling to study the metabolic turnover of adenylate cyclase in a nonfusing muscle cell line, the BC/sup 3/H1 cells. These cells contains an adenylate cyclase coupled to beta-adrenergic receptors and highly stimulated by forskolin, a potent activator of the enzyme. After transfer of the cells from normal medium to heavy medium (a medium containing heavy labeled amino acids, /sup 3/H, /sup 13/C, /sup 15/N), heavy isotope-labeled adenylate cyclase molecules progressively replace pre-existing light molecules. In sucrose gradient differential sedimentation, after a 5-day switch in heavy medium, the enzyme exhibited a higher massmore » (s = 8.40 +/- 0.03 S, n = 13) compared to the control enzyme. Indeed, the increase in the sedimentation coefficient of the heavy molecules was due to the synthesis of new molecules of adenylate cyclase labeled with heavy isotope amino acids since in the presence of cycloheximide, an inhibitor of protein synthesis, no change in the sedimentation pattern of the forskolin-stimulated adenylate cyclase occurred. After incorporation of heavy isotope amino acids in the adenylate cyclase molecules, the kinetics parameters of the enzyme did not change. However, adenylate cyclase from cells incubated with heavy medium exhibits an activity about 2-fold lower than control. After switching the cells to the heavy medium, the decrease of the activity of the enzyme occurred during the first 24 h and thereafter remained at a steady state for at least 4 days. In contrast, 24 h after the switch, the sedimentation coefficient of forskolin-stimulated adenylate cyclase was progressively shifted to a higher value.« less

  14. The effects of the soluble guanylate cyclase stimulator riociguat on memory performance in healthy volunteers with a biperiden-induced memory impairment.

    PubMed

    Borghans, Laura G J M; Sambeth, Anke; Prickaerts, Jos; Ramaekers, Johannes G; Blokland, Arjan

    2018-06-07

    After stimulation with nitric oxide, soluble guanylate cyclase (sGC) produces cyclic guanosine monophosphate (cGMP), which stimulates an important signalling pathway for long-term potentiation (LTP). By upregulating cGMP, LTP could be stimulated and thereby enhancing memory processes. The present study investigated the effects of the sGC stimulator riociguat on cognition in healthy volunteers. Participants were pre-treated with and without biperiden, which impairs memory performance, to investigate the memory-enhancing effects of riociguat. Twenty volunteers participated in a double-blind placebo-controlled six-way crossover design with a cognitive test battery including the verbal learning task (VLT), n-back task, spatial memory test, the attention network test, and a reaction time task. Treatments were placebo and riociguat 0.5 mg, placebo and riociguat 1.0 mg, biperiden 2.0 mg and placebo, biperiden 2.0 mg and riociguat 0.5 mg and biperiden 2.0 mg and riociguat 1.0 mg. Blood pressure was found to be decreased and heart rate to be increased after administration of riociguat. Cognitive performance was not enhanced after administration of riociguat. Biperiden decreased episodic memory on the VLT, yet this deficit was not reversed by riociguat. This supports the notion that biperiden might be a valuable pharmacological model to induce episodic memory impairments as observed in AD/MCI.

  15. Extracellular cAMP activates molecular signalling pathways associated with sperm capacitation in bovines.

    PubMed

    Alonso, Carlos Agustín I; Osycka-Salut, Claudia E; Castellano, Luciana; Cesari, Andreína; Di Siervi, Nicolás; Mutto, Adrián; Johannisson, Anders; Morrell, Jane M; Davio, Carlos; Perez-Martinez, Silvina

    2017-08-01

    Is extracellular cAMP involved in the regulation of signalling pathways in bovine sperm capacitation? Extracellular cAMP induces sperm capacitation through the activation of different signalling pathways that involve phospholipase C (PLC), PKC/ERK1-2 signalling and an increase in sperm Ca2+ levels, as well as soluble AC and cAMP/protein kinase A (PKA) signalling. In order to fertilize the oocyte, ejaculated spermatozoa must undergo a series of changes in the female reproductive tract, known as capacitation. This correlates with a number of membrane and metabolic modifications that include an increased influx of bicarbonate and Ca2+, activation of a soluble adenylyl cyclase (sAC) to produce cAMP, PKA activation, protein tyrosine phosphorylation and the development of hyperactivated motility. We previously reported that cAMP efflux by Multidrug Resistance Protein 4 (MRP4) occurs during sperm capacitation and the pharmacological blockade of this inhibits the process. Moreover, the supplementation of incubation media with cAMP abolishes the inhibition and leads to sperm capacitation, suggesting that extracellular cAMP regulates crucial signalling cascades involved in this process. Bovine sperm were selected by the wool glass column method, and washed by centrifugation in BSA-Free Tyrode's Albumin Lactate Pyruvate (sp-TALP). Pellets were resuspended then diluted for each treatment. For in vitro capacitation, 10 to 15 × 106 SPZ/ml were incubated in 0.3% BSA sp-TALP at 38.5°C for 45 min under different experimental conditions. To evaluate the role of extracellular cAMP on different events associated with sperm capacitation, 10 nM cAMP was added to the incubation medium as well as different inhibitors of enzymes associated with signalling transduction pathways: U73122 (PLC inhibitor, 10 μM), Gö6983 (PKC inhibitor, 10 μM), PD98059 (ERK-1/2 inhibitor, 30 μM), H89 and KT (PKA inhibitors, 50 μM and 100 nM, respectively), KH7 (sAC inhibitor, 10 μM), BAPTA

  16. Isolation and functional characterization of Lycopene β-cyclase (CYC-B) promoter from Solanum habrochaites

    PubMed Central

    2010-01-01

    Background Carotenoids are a group of C40 isoprenoid molecules that play diverse biological and ecological roles in plants. Tomato is an important vegetable in human diet and provides the vitamin A precursor β-carotene. Genes encoding enzymes involved in carotenoid biosynthetic pathway have been cloned. However, regulation of genes involved in carotenoid biosynthetic pathway and accumulation of specific carotenoid in chromoplasts are not well understood. One of the approaches to understand regulation of carotenoid metabolism is to characterize the promoters of genes encoding proteins involved in carotenoid metabolism. Lycopene β-cyclase is one of the crucial enzymes in carotenoid biosynthesis pathway in plants. Its activity is required for synthesis of both α-and β-carotenes that are further converted into other carotenoids such as lutein, zeaxanthin, etc. This study describes the isolation and characterization of chromoplast-specific Lycopene β-cyclase (CYC-B) promoter from a green fruited S. habrochaites genotype EC520061. Results A 908 bp region upstream to the initiation codon of the Lycopene β-cyclase gene was cloned and identified as full-length promoter. To identify promoter region necessary for regulating developmental expression of the ShCYC-B gene, the full-length promoter and its three different 5' truncated fragments were cloned upstream to the initiation codon of GUS reporter cDNA in binary vectors. These four plant transformation vectors were separately transformed in to Agrobacterium. Agrobacterium-mediated transient and stable expression systems were used to study the GUS expression driven by the full-length promoter and its 5' deletion fragments in tomato. The full-length promoter showed a basal level activity in leaves, and its expression was upregulated > 5-fold in flowers and fruits in transgenic tomato plants. Deletion of -908 to -577 bp 5' to ATG decreases the ShCYC-B promoter strength, while deletion of -908 to -437 bp 5' to ATG led to

  17. Redesign of Schistosoma mansoni NAD+ catabolizing enzyme : the active site H103W mutation restores ADP-ribosyl cyclase activity†

    PubMed Central

    Kuhn, Isabelle; Kellenberger, Esther; Rognan, Didier; Lund, Frances E.; Muller-Steffner, Hélène; Schuber, Francis

    2008-01-01

    Schistosoma mansoni NAD(P)+ catabolizing enzyme (SmNACE) is a new member of the ADP-ribosyl cyclase family. In contrast to all the other enzymes which are involved in the production of metabolites that elicit Ca2+ mobilization, SmNACE is virtually unable to transform NAD+ into the second messenger cyclic ADP-ribose (cADPR). Sequence alignments revealed that one of four conserved residues within the active site of these enzymes was replaced in SmNACE by a histidine (His103) instead of the highly conserved tryptophan. To find out whether the inability of SmNACE to catalyze the canonical ADP-ribosyl cyclase reaction is linked to this change we have replaced His103 with a tryptophan. The H103W mutation in SmNACE was indeed found to restore ADP-ribosyl cyclase activity as cADPR amounts for 7% of the reaction products, i.e., a value larger than observed for other members of this family such as CD38. Introduction of a Trp103 residue provides some of the binding characteristics of mammalian ADP-ribosyl cyclases such as increased affinity for Cibacron blue and slow-binding inhibition by araF-NAD+. Homology modeling of wild-type and H103W mutant three-dimensional structures, and docking of substrates within the active sites, provide new insight into the catalytic mechanism of SmNACE. Both residue side chains share similar roles in the nicotinamide-ribose bond cleavage step leading to an E.ADP-ribosyl reaction intermediate. They diverge however in the evolution of this intermediate; His103 provides a more polar environment favoring the accessibility to water and hydrolysis leading to ADP-ribose at the expense of the intramolecular cyclization pathway resulting in cADPR. PMID:17002287

  18. Phenylalanine 445 within oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae influences C-Ring cyclization and deprotonation reactions.

    PubMed

    Wu, Tung-Kung; Liu, Yuan-Ting; Chiu, Feng-Hsuan; Chang, Cheng-Hsiang

    2006-10-12

    [reaction: see text] We describe the Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase Phe445 site-saturated mutants that generate truncated tricyclic and altered deprotonation product profiles. Among these mutants, only polar side-chain group substitutions genetically complemented yeast viability and produced spatially related product diversity, supporting the Johnson model that cation-pi interactions between a carbocationic intermediate and an enzyme can be replaced by an electrostatic or polar side chain to stabilize the cationic intermediate, but with product differentiation.

  19. Forskolin- and dihydroalprenolol (DHA) binding sites and adenylate cyclase activity in heart of rats fed diets containing different oils

    SciTech Connect

    Alam, S.Q.; Ren, Y.F.; Alam, B.S.

    1987-05-01

    The purpose of the present investigation was to determine if dietary lipids can induce changes in the adenylate cyclase system in rat heart. Three groups of male young Sprague-Dawley rats were fed for 6 weeks diets containing 10% corn oil (I), 8% coconut oil + 2% corn oil (II) or 10% menhaden oil (III). Adenylate cyclase activity (basal, fluoride-, isoproterenol-, and forskolin-stimulated) was higher in heart homogenates of rats in group III than in the other two groups. Concentration of the (/sup 3/H)-forskolin binding sites in the cardiac membranes were significantly higher in rats fed menhaden oil. The values (pmol/mgmore » protein) were 4.8 +/- 0.2 (I), 4.5 +/- 0.7 (II) and 8.4 +/- 0.5 (III). There was no significant difference in the affinity of the forskolin binding sites among the 3 dietary groups. When measured at different concentrations of forskolin, the adenylate cyclase activity in cardiac membranes of rats fed menhaden oil was higher than in the other 2 groups. Concentrations of the (/sup 3/H)DHA binding sites were slightly higher but their affinity was lower in cardiac membranes of rats fed menhaden oil. The results suggest that diets containing fish oil increase the concentration of the forskolin binding sites and may also affect the characteristics of the ..beta..-adrenergic receptor in rat heart.« less

  20. Characterization of a novel serotonin receptor coupled to adenylate cyclase in the hybrid neuroblastoma cell line NCB. 20

    SciTech Connect

    Conner, D.A.

    1988-01-01

    Pharmacological characterization of the serotonin activation of adenylate cyclase in membrane preparation using over 40 serotonergic and non-serotonergic compounds demonstrated that the receptor mediating the response was distinct from previously described mammalian serotonin receptors. Agonist activity was only observed with tryptamine and ergoline derivatives. Potent antagonism was observed with several ergoline derivatives and with compounds such as mianserin and methiothepine. A comparison of the rank order of potency of a variety of compounds for the NCB.20 cell receptor with well characterized mammalian and non-mammalian serotonin receptors showed a pharmacological similarity, but not identity, with the mammalian 5-HT{sub 1C} receptor, whichmore » modulates phosphatidylinositol metabolism, and with serotonin receptors in the parasitic trematodes Fasciola hepatica and Schistosoma mansoni, which are coupled to adenylate cyclase. Equilibrium binding analysis utilizing ({sup 3}H)serotonin, ({sup 3}H)lysergic acid diethylamide or ({sup 3}H)dihydroergotamine demonstrated that there are no abundant high affinity serotonergic sites, which implies that the serotonin activation of adenylate cyclase is mediated by receptors present in low abundance. Incubation of intact NCB.20 cells with serotinin resulted in a time and concentration dependent desensitization of the serotonin receptor.« less

  1. Localization of the action of cholera toxin on adenyl cyclase in mucosal epithelial cells of rabbit intestine

    PubMed Central

    Parkinson, David K.; Ebel, Hans; DiBona, Donald R.; Sharp, Geoffrey W. G.

    1972-01-01

    Brush borders and plasma membranes have been purified from mucosal epithelial cells of rabbit ileum under control conditions and after treatment for 3 hr with cholera toxin in vivo. The activity of several enzymes in these preparations was measured. It was concluded that adenyl cyclase, like NaK-ATPase, seems not to be a normal constituent of brush borders. Both these enzymes are present in plasma membrane preparations derived largely from the basal and lateral margins of the epithelial cells, both may be phospholipid dependent enzymes and both are affected by cholera toxin. Adenyl cyclase activity is increased while NaK-ATPase is decreased. The activities of alkaline phosphatase, leucineaminopeptidase, 5′-nucleotidase, glucose-6-phosphatase, and Mg-ATPase were not found to be affected by the toxin. Cholera toxin, which makes contact with the luminal side of the epithelial cells, in the natural disease and in the experimental model, would appear to exert its pathologic effect on adenyl cyclase at the opposite (basal and lateral) side of the cells. Images PMID:4344729

  2. Diversity of squalene-hopene cyclases in a tropical carbonate-rich environment

    NASA Astrophysics Data System (ADS)

    Leavitt, W. D.; Pearson, A.

    2007-12-01

    Hopanoids are isoprenoid lipids which derive primarily from bacteria and are ubiquitous in contemporary Earth surface environments. In the geologic record, hopanes found in sedimentary rocks are used as proxies to help decipher ancient biological communities. However, in contrast to the ubiquity of these lipid products, biosynthesis of hopanoids appears to be a relatively rare physiological trait among bacteria in complex environmental communities. We have recently estimated that fewer than one in ten bacterial cells in soils and fewer than one in twenty bacterial cells in the ocean contains the gene squalene-hopene cyclase (sqhC) [1]. Biosynthesis of hopanoids is rarer in natural communities than it is among species that have been propagated in pure culture [2]. Here we continue our previous work to survey the phylogeny and diversity of hopanoid producers using culture-independent methods. In particular, genes affiliated with known cyanobacterial sequences were not detected in the contemporary environments analyzed previously [1]. One possible explanation is that hopanoid-producing strains of cyanobacteria are regionally localized. It has been suggested that throughout the long-term sedimentary record there is a correlation between 2-methylhopanoid index (a putative indicator of cyanobacterial biomass) and the global prevalence of shallow carbonate platform environments [3], and in previous work we did not analyze any such environments. To address this question we surveyed a land-sea gradient across the Bahamian island of San Salvador. Samples were taken from upland soil, a hypersaline lake, a tidal creek, and the shallow open ocean. The data are remarkably similar to our previous results: environmental sqhCs average < 65% translated amino acid identity to their closest relatives in public databases, and non- cyanobacterial sequences continue to dominate. We will discuss the challenges these results pose for deciphering the global distribution of microbially

  3. Further investigation into the signal transduction mechanism of the 5-HT4-like receptor in the circular smooth muscle of human colon.

    PubMed Central

    McLean, P. G.; Coupar, I. M.

    1996-01-01

    1. The nature of the receptor coupling mechanism of the 5-hydroxytryptamine4 (5-HT4) receptor in the circular smooth muscle of the human colon has been further investigated. 2. 5-HT stimulated cyclic AMP generation and caused a relaxation in a concentration-dependent fashion, with EC50 values of 175.5 and 274.9 nM respectively. DAU 6236 increased cyclic AMP formation and caused a relaxant effect but was a partial agonist relative to 5-HT. 3. The 5-HT4 receptor antagonist, GR 113808, inhibited cyclic AMP formation and relaxation induced by 5-HT with -log Ki values of 9.1 (cyclic AMP) and 8.9 (relaxation) and apparent pA2 values of 9.2 (cyclic AMP) and 9.5 (relaxation). 4. Ondansetron and methysergide failed to inhibit cyclic AMP formation or the relaxation induced by 5-HT. 5. The phosphodiesterase inhibitor, IBMX, produced a concentration-dependent relaxation (EC50 = 30 microM) and at 1 microM it enhanced the 5-HT-induced relaxation producing a leftward shift of the 5-HT concentration-effect curve with a concentration-ratio of 4.1. Rolipram caused a concentration-dependent relaxation (EC50 = 564.8 nM) and at 200 nm caused a leftward shift of the concentration-effect curve to 5-HT with a concentration-ratio of 5.5. 6. Application of the adenylyl cyclase inhibitor, SQ 22536 (0.1 mM), and the protein kinase inhibitors, H7 (100 nM) and H89 (200 nM), inhibited the relaxant effect of 5-HT inducing a rightward shift of the concentration-effect curve with concentration-ratios of 10.1, 2.7 and 4.2 respectively. 7. Forskolin stimulated cyclic AMP production and caused a relaxation. The maximum relaxant effect of forskolin (6 microM, 13.8 +/- 1.9 cm.s) was not significantly different from the maximum relaxant effect of 5-HT (10 microM, 12.7 +/- 4.9 cm.s). However, the cyclic AMP levels stimulated by forskolin (6 microM, 49.3 +/- 6.6 pmol mg-1) were markedly greater than those stimulated by 5-HT (10 microM, 7.6 +/- 2.0 pmol mg-1). 8. In conclusion, these results indicate that

  4. Further investigation into the signal transduction mechanism of the 5-HT4-like receptor in the circular smooth muscle of human colon.

    PubMed

    McLean, P G; Coupar, I M

    1996-06-01

    1. The nature of the receptor coupling mechanism of the 5-hydroxytryptamine4 (5-HT4) receptor in the circular smooth muscle of the human colon has been further investigated. 2. 5-HT stimulated cyclic AMP generation and caused a relaxation in a concentration-dependent fashion, with EC50 values of 175.5 and 274.9 nM respectively. DAU 6236 increased cyclic AMP formation and caused a relaxant effect but was a partial agonist relative to 5-HT. 3. The 5-HT4 receptor antagonist, GR 113808, inhibited cyclic AMP formation and relaxation induced by 5-HT with -log Ki values of 9.1 (cyclic AMP) and 8.9 (relaxation) and apparent pA2 values of 9.2 (cyclic AMP) and 9.5 (relaxation). 4. Ondansetron and methysergide failed to inhibit cyclic AMP formation or the relaxation induced by 5-HT. 5. The phosphodiesterase inhibitor, IBMX, produced a concentration-dependent relaxation (EC50 = 30 microM) and at 1 microM it enhanced the 5-HT-induced relaxation producing a leftward shift of the 5-HT concentration-effect curve with a concentration-ratio of 4.1. Rolipram caused a concentration-dependent relaxation (EC50 = 564.8 nM) and at 200 nm caused a leftward shift of the concentration-effect curve to 5-HT with a concentration-ratio of 5.5. 6. Application of the adenylyl cyclase inhibitor, SQ 22536 (0.1 mM), and the protein kinase inhibitors, H7 (100 nM) and H89 (200 nM), inhibited the relaxant effect of 5-HT inducing a rightward shift of the concentration-effect curve with concentration-ratios of 10.1, 2.7 and 4.2 respectively. 7. Forskolin stimulated cyclic AMP production and caused a relaxation. The maximum relaxant effect of forskolin (6 microM, 13.8 +/- 1.9 cm.s) was not significantly different from the maximum relaxant effect of 5-HT (10 microM, 12.7 +/- 4.9 cm.s). However, the cyclic AMP levels stimulated by forskolin (6 microM, 49.3 +/- 6.6 pmol mg-1) were markedly greater than those stimulated by 5-HT (10 microM, 7.6 +/- 2.0 pmol mg-1). 8. In conclusion, these results indicate that

  5. Soluble guanylyl cyclase-activated cyclic GMP-dependent protein kinase inhibits arterial smooth muscle cell migration independent of VASP-serine 239 phosphorylation.

    PubMed

    Holt, Andrew W; Martin, Danielle N; Shaver, Patti R; Adderley, Shaquria P; Stone, Joshua D; Joshi, Chintamani N; Francisco, Jake T; Lust, Robert M; Weidner, Douglas A; Shewchuk, Brian M; Tulis, David A

    2016-09-01

    Coronary artery disease (CAD) accounts for over half of all cardiovascular disease-related deaths. Uncontrolled arterial smooth muscle (ASM) cell migration is a major component of CAD pathogenesis and efforts aimed at attenuating its progression are clinically essential. Cyclic nucleotide signaling has long been studied for its growth-mitigating properties in the setting of CAD and other vascular disorders. Heme-containing soluble guanylyl cyclase (sGC) synthesizes cyclic guanosine monophosphate (cGMP) and maintains vascular homeostasis predominantly through cGMP-dependent protein kinase (PKG) signaling. Considering that reactive oxygen species (ROS) can interfere with appropriate sGC signaling by oxidizing the cyclase heme moiety and so are associated with several CVD pathologies, the current study was designed to test the hypothesis that heme-independent sGC activation by BAY 60-2770 (BAY60) maintains cGMP levels despite heme oxidation and inhibits ASM cell migration through phosphorylation of the PKG target and actin-binding vasodilator-stimulated phosphoprotein (VASP). First, using the heme oxidant ODQ, cGMP content was potentiated in the presence of BAY60. Using a rat model of arterial growth, BAY60 significantly reduced neointima formation and luminal narrowing compared to vehicle (VEH)-treated controls. In rat ASM cells BAY60 significantly attenuated cell migration, reduced G:F actin, and increased PKG activity and VASP Ser239 phosphorylation (pVASP·S239) compared to VEH controls. Site-directed mutagenesis was then used to generate overexpressing full-length wild type VASP (FL-VASP/WT), VASP Ser239 phosphorylation-mimetic (FL-VASP/239D) and VASP Ser239 phosphorylation-resistant (FL-VASP/239A) ASM cell mutants. Surprisingly, FL-VASP/239D negated the inhibitory effects of FL-VASP/WT and FL-VASP/239A cells on migration. Furthermore, when FL-VASP mutants were treated with BAY60, only the FL-VASP/239D group showed reduced migration compared to its VEH controls

  6. Functional Lycopene Cyclase (CruA) in Cyanobacterium, Arthrospira platensis NIES-39, and its Role in Carotenoid Synthesis.

    PubMed

    Sugiyama, Kenjiro; Ebisawa, Masashi; Yamada, Masaharu; Nagashima, Yoshiki; Suzuki, Hideyuki; Maoka, Takashi; Takaichi, Shinichi

    2017-04-01

    The genus Arthrospira is filamentous, non-nitrogen-fixing cyanobacteria that is commercially important. We identified the molecular structures of carotenoids in Arthrospira platensis NIES-39. The major carotenoid identified was β-carotene. In addition, the hydroxyl derivatives of β-cryptoxanthin and (3R,3'R)-zeaxanthin were also found to be present. The carotenoid glycosides were identified as (3R,2'S)-myxol 2'-methylpentoside and oscillol 2,2'-dimethylpentoside. The methylpentoside moiety was a mixture of fucoside and chinovoside in an approximate ratio of 1 : 4. Trace amounts of the ketocarotenoid 3'-hydroxyechinenone were also found. Three types of lycopene cyclases have been functionally confirmed in carotenogenesis organisms. In cyanobacteria, the functional lycopene cyclases (CrtL, CruA and CruP) have only been found in four species. In this study, we found that CruA exhibited lycopene cyclase activity in transformed Escherichia coli, which contains lycopene, but CruP exhibited no lycopene cyclase activity and crtL was absent. This is the third cyanobacterial species in which CruA activity has been confirmed. Neurosporene was not a substrate of CruA in E. coli, whereas lycopene cyclases of CrtY (bacteria), CrtL (plants) and CrtYB (fungi) have been reported to convert neurosporene to 7,8-dihydro-β-carotene. β-Carotene hydroxylase (CrtR) was found to convert β-carotene to zeaxanthin in transformed E. coli, which contains β-carotene. Among the β-carotene hydroxylases, bacterial CrtZ and eukaryotic CrtR and BCH have similarities, whereas cyanobacterial CrtR appears to belong to another clade. Based on the identification of the carotenoids and the completion of the entire nucleotide sequence of the A. platensis NIES-39 genome, we propose a biosynthetic pathway for the carotenoids as well as the corresponding genes and enzymes. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved

  7. Disaggregation of adenylate cyclase during polyacrylamide-gel electrophoresis in mixtures of ionic and non-ionic detergents.

    PubMed

    Newby, A C; Chrambach, A

    1979-02-01

    1. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] solubilized from the rat liver plasma membrane with 1% Lubrol PX and partially purified by gel filtration in buffer containing 0.01% Lubrol PX was physically characterized by polyacrylamide-gel electrophoresis. 2. The molecular radius determined for the partially purified enzyme was 4.9nm, compared with the value of 3.9nm obtained for the enzyme before gel filtration. 3. This difference, representing an approximate doubling of the molecular volume of the enzyme, implied that aggregation with itself or other proteins had occurred during partial purification. 4. Aggregation was not reversed by electrophoresis in the presence of high Lubrol concentrations. 5. Substitution of deoxycholate or N-dodecylsarcosinate for Lubrol PX either for solubilization or during electrophoresis led to poorer resolution of membrane proteins at concentrations giving greater than 70% loss of enzyme activity. 6. Partially purified adenylate cyclase was electrophoresed in the presence of mixed micelles of Lubrol PX and deoxycholate or Lubrol PX and N-dodecylsarcosinate. Different mixtures were examined simultaneously in a suitable apparatus. 7. Electrophoresis in the presence of 0.1% Lubrol plus 0.03% deoxycholate decreased the molecular radius of the cyclase to 4.0nm, with greater than 90% recovery of enzymic activity. The net charge of the enzyme was also increased, indicating ionic detergent binding. 8. With 0.1% Lubrol plus 0.03% N-dodecylsarcosinate the molecular radius was 4.3nm, recovery approx. 50% and net charge similar to that seen in Lubrol plus deoxycholate. 9. The resolution of cyclase from bulk protein, on an analytical scale, was improved in the presence of detergent mixtures, as compared with resolution in Lubrol alone. 10. The results demonstrate the usefulness of polyacrylamide-gel electrophoresis to detect and overcome aggregation problems with membrane proteins and suggest that detergent mixtures in

  8. Disaggregation of adenylate cyclase during polyacrylamide-gel electrophoresis in mixtures of ionic and non-ionic detergents

    PubMed Central

    Newby, Andrew C.; Chrambach, Andreas

    1979-01-01

    1. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] solubilized from the rat liver plasma membrane with 1% Lubrol PX and partially purified by gel filtration in buffer containing 0.01% Lubrol PX was physically characterized by polyacrylamide-gel electrophoresis. 2. The molecular radius determined for the partially purified enzyme was 4.9nm, compared with the value of 3.9nm obtained for the enzyme before gel filtration. 3. This difference, representing an approximate doubling of the molecular volume of the enzyme, implied that aggregation with itself or other proteins had occurred during partial purification. 4. Aggregation was not reversed by electrophoresis in the presence of high Lubrol concentrations. 5. Substitution of deoxycholate or N-dodecylsarcosinate for Lubrol PX either for solubilization or during electrophoresis led to poorer resolution of membrane proteins at concentrations giving greater than 70% loss of enzyme activity. 6. Partially purified adenylate cyclase was electrophoresed in the presence of mixed micelles of Lubrol PX and deoxycholate or Lubrol PX and N-dodecylsarcosinate. Different mixtures were examined simultaneously in a suitable apparatus. 7. Electrophoresis in the presence of 0.1% Lubrol plus 0.03% deoxycholate decreased the molecular radius of the cyclase to 4.0nm, with greater than 90% recovery of enzymic activity. The net charge of the enzyme was also increased, indicating ionic detergent binding. 8. With 0.1% Lubrol plus 0.03% N-dodecylsarcosinate the molecular radius was 4.3nm, recovery approx. 50% and net charge similar to that seen in Lubrol plus deoxycholate. 9. The resolution of cyclase from bulk protein, on an analytical scale, was improved in the presence of detergent mixtures, as compared with resolution in Lubrol alone. 10. The results demonstrate the usefulness of polyacrylamide-gel electrophoresis to detect and overcome aggregation problems with membrane proteins and suggest that detergent mixtures in

  9. Pituitary adenylate cyclase-activating polypeptide is a potent inhibitor of the growth of light chain-secreting human multiple myeloma cells.

    PubMed

    Li, Min; Cortez, Shirley; Nakamachi, Tomoya; Batuman, Vecihi; Arimura, Akira

    2006-09-01

    Multiple myeloma represents a malignant proliferation of plasma cells in the bone marrow, which often overproduces immunoglobulin light chains. We have shown previously that pituitary adenylate cyclase-activating polypeptide (PACAP) markedly suppresses the release of proinflammatory cytokines from light chain-stimulated human renal proximal tubule epithelial cells and prevents the resulting tubule cell injury. In this study, we have shown that PACAP suppresses the proliferation of human kappa and lambda light chain-secreting multiple myeloma-derived cells. The addition of PACAP suppressed light chain-producing myeloma cell-stimulated interleukin 6 (IL-6) secretion by the bone marrow stromal cells (BMSCs). A specific antagonist to either the human PACAP-specific receptor or the vasoactive intestinal peptide receptor attenuated the suppressive effect of PACAP on IL-6 production in the adhesion of human multiple myeloma cells to BMSCs. The secretion of IL-6 by BMSCs was completely inhibited by 10(-9) mol/L PACAP, which also attenuated the phosphorylation of both p42/44 and p38 mitogen-activated protein kinases (MAPK) as well as nuclear factor-kappaB (NF-kappaB) activation in response to the adhesion of multiple myeloma cells to BMSCs, whereas the inhibition of p42/44 MAPK signaling attenuated PACAP action. The signaling cascades involved in the inhibitory effect of PACAP on IL-6-mediated paracrine stimulation of light chain-secreting myeloma cell growth was mediated through the suppression of p38 MAPK as well as modulation of activation of transcription factor NF-kappaB. These findings suggest that PACAP may be a new antitumor agent that directly suppresses light chain-secreting myeloma cell growth and indirectly affects tumor cell growth by modifying the bone marrow milieu of the multiple myeloma.

  10. A membrane-associated adenylate cyclase modulates lactate dehydrogenase and creatine kinase activities required for bull sperm capacitation induced by hyaluronic acid.

    PubMed

    Fernández, Silvina; Córdoba, Mariana

    2017-04-01

    Hyaluronic acid, as well as heparin, is a glycosaminoglycan present in the female genital tract of cattle. The aim of this study was to evaluate oxidative metabolism and intracellular signals mediated by a membrane-associated adenylate cyclase (mAC), in sperm capacitation with hyaluronic acid and heparin, in cryopreserved bull sperm. The mAC inhibitor, 2',5'-dideoxyadenosine, was used in the present study. Lactate dehydrogenase (LDH) and creatine kinase (CK) activities and lactate concentration were determined spectrophotometrically in the incubation medium. Capacitation and acrosome reaction were evaluated by chlortetracycline technique, while plasma membrane and acrosome integrity were determined by trypan blue stain/differential interference contrast microscopy. Heparin capacitated samples had a significant decrease in LDH and CK activities, while in hyaluronic acid capacitated samples LDH and CK activities both increased compared to control samples, in heparin and hyaluronic acid capacitation conditions, respectively. A significant increase in lactate concentration in the incubation medium occurred in hyaluronic acid-treated sperm samples compared to heparin treatment, indicating this energetic metabolite is produced during capacitation. The LDH and CK enzyme activities and lactate concentrations in the incubation medium were decreased with 2',5'-dideoxyadenosine treatment in hyaluronic acid samples. The mAC inhibitor significantly inhibited heparin-induced capacitation of sperm cells, but did not completely inhibit hyaluronic acid capacitation. Therefore, hyaluronic acid and heparin are physiological glycosaminoglycans capable of inducing in vitro capacitation in cryopreserved bull sperm, stimulating different enzymatic pathways and intracellular signals modulated by a mAC. Hyaluronic acid induces sperm capacitation involving LDH and CK activities, thereby reducing oxidative metabolism, and this process is mediated by mAC. Copyright © 2017 Elsevier B.V. All

  11. Nitric oxide in B6 mouse and nitric oxide-sensitive soluble guanylate cyclase in cat modulate acetylcholine release in pontine reticular formation.

    PubMed

    Lydic, Ralph; Garza-Grande, Ricardo; Struthers, Richard; Baghdoyan, Helen A

    2006-05-01

    ACh regulates arousal, and the present study was designed to provide insight into the neurochemical mechanisms modulating ACh release in the pontine reticular formation. Nitric oxide (NO)-releasing beads microinjected into the pontine reticular formation of C57BL/6J (B6) mice significantly (P < 0.0001) increased ACh release. Microdialysis delivery of the NO donor N-ethyl-2-(1-ethyl-2-hydroxy-2-nitrosohydrazino)-ethanamine (NOC-12) to the mouse pontine reticular formation also caused a concentration-dependent increase in ACh release (P < 0.001). These are the first neurochemical data showing that ACh release in the pontine reticular formation of the B6 mouse is modulated by NO. The signal transduction cascade through which NO modulates ACh release in the pontine reticular formation has not previously been characterized. Therefore, an additional series of studies quantified the effects of a soluble guanylate cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), on ACh release in the cat medial pontine reticular formation. During naturally occurring states of sleep and wakefulness, but not anesthesia, ODQ caused a significant (P < 0.001) decrease in ACh release. These results show for the first time that NO modulates ACh in the medial pontine reticular formation of the cat via an NO-sensitive sGC signal transduction cascade. Isoflurane and halothane anesthesia have been shown to decrease ACh release in the medial pontine reticular formation. The finding that ODQ did not alter ACh release during isoflurane or halothane anesthesia demonstrates that these anesthetics disrupt the NO-sensitive sGC-cGMP pathway. Considered together, results from the mouse and cat indicate that NO modulates ACh release in arousal-promoting regions of the pontine reticular formation via an NO-sensitive sGC-cGMP pathway.

  12. Overexpression of adenylate cyclase-associated protein 2 is a novel prognostic marker in malignant melanoma.

    PubMed

    Masugi, Yohei; Tanese, Keiji; Emoto, Katsura; Yamazaki, Ken; Effendi, Kathryn; Funakoshi, Takeru; Mori, Mariko; Sakamoto, Michiie

    2015-12-01

    Malignant melanoma is one of the lethal malignant tumors worldwide. Previously we reported that adenylate cyclase-associated protein 2 (CAP2), which is a well-conserved actin regulator, was overexpressed in hepatocellular carcinoma; however, CAP2 expression in other clinical cancers remains unclear. The aim of the current study was to clarify the clinicopathological significance of CAP2 overexpression in malignant melanoma. Immunohistochemical analyses revealed that many melanoma cells exhibited diffuse cytoplasmic expression of CAP2, whereas no normal melanocytes showed detectable immunostaining for CAP2. A high level of CAP2 expression was seen in 14 of 50 melanomas and was significantly correlated with greater tumor thickness and nodular melanoma subtypes. In addition, a high level of CAP2 expression was associated with poor overall survival in univariate and multivariate analyses. For 13 patients, samples of primary and metastatic melanoma tissue were available: four patients exhibited higher levels of CAP2 expression in metastatic tumor compared to the primary site, whereas no patient showed lower levels of CAP2 expression in metastatic melanomas. Our findings show that CAP2 overexpression is a novel prognostic marker in malignant melanoma and that CAP2 expression seems to increase stepwise during tumor progression, suggesting the involvement of CAP2 in the aggressive behavior of malignant melanoma. © 2015 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.

  13. Levels of Lycopene β-Cyclase 1 Modulate Carotenoid Gene Expression and Accumulation in Daucus carota

    PubMed Central

    Moreno, Juan Camilo; Pizarro, Lorena; Fuentes, Paulina; Handford, Michael; Cifuentes, Victor; Stange, Claudia

    2013-01-01

    Plant carotenoids are synthesized and accumulated in plastids through a highly regulated pathway. Lycopene β-cyclase (LCYB) is a key enzyme involved directly in the synthesis of α-carotene and β-carotene through the cyclization of lycopene. Carotenoids are produced in both carrot (Daucus carota) leaves and reserve roots, and high amounts of α-carotene and β-carotene accumulate in the latter. In some plant models, the presence of different isoforms of carotenogenic genes is associated with an organ-specific function. D. carota harbors two Lcyb genes, of which DcLcyb1 is expressed in leaves and storage roots during carrot development, correlating with an increase in carotenoid levels. In this work, we show that DcLCYB1 is localized in the plastid and that it is a functional enzyme, as demonstrated by heterologous complementation in Escherichia coli and over expression and post transcriptional gene silencing in carrot. Transgenic plants with higher or reduced levels of DcLcyb1 had incremented or reduced levels of chlorophyll, total carotenoids and β-carotene in leaves and in the storage roots, respectively. In addition, changes in the expression of DcLcyb1 are accompanied by a modulation in the expression of key endogenous carotenogenic genes. Our results indicate that DcLcyb1 does not possess an organ specific function and modulate carotenoid gene expression and accumulation in carrot leaves and storage roots. PMID:23555569

  14. Pituitary Adenylate Cyclase-Activating Polypeptide Reverses Ammonium Metavanadate-Induced Airway Hyperresponsiveness in Rats

    PubMed Central

    Tlili, Mounira; Sriha, Badreddine; Ben Rhouma, Khémais; Sakly, Mohsen; Wurtz, Olivier

    2015-01-01

    The rate of atmospheric vanadium is constantly increasing due to fossil fuel combustion. This environmental pollution favours vanadium exposure in particular to its vanadate form, causing occupational bronchial asthma and bronchitis. Based on the well admitted bronchodilator properties of the pituitary adenylate cyclase-activating polypeptide (PACAP), we investigated the ability of this neuropeptide to reverse the vanadate-induced airway hyperresponsiveness in rats. Exposure to ammonium metavanadate aerosols (5 mg/m3/h) for 15 minutes induced 4 hours later an array of pathophysiological events, including increase of bronchial resistance and histological alterations, activation of proinflammatory alveolar macrophages, and increased oxidative stress status. Powerfully, PACAP inhalation (0.1 mM) for 10 minutes alleviated many of these deleterious effects as demonstrated by a decrease of bronchial resistance and histological restoration. PACAP reduced the level of expression of mRNA encoding inflammatory chemokines (MIP-1α, MIP-2, and KC) and cytokines (IL-1α and TNF-α) in alveolar macrophages and improved the antioxidant status. PACAP reverses the vanadate-induced airway hyperresponsiveness not only through its bronchodilator activity but also by counteracting the proinflammatory and prooxidative effects of the metal. Then, the development of stable analogs of PACAP could represent a promising therapeutic alternative for the treatment of inflammatory respiratory disorders. PMID:26199679

  15. Intestinal Cell Proliferation and Senescence Are Regulated by Receptor Guanylyl Cyclase C and p21*

    PubMed Central

    Basu, Nirmalya; Saha, Sayanti; Khan, Imran; Ramachandra, Subbaraya G.; Visweswariah, Sandhya S.

    2014-01-01

    Guanylyl cyclase C (GC-C) is expressed in intestinal epithelial cells and serves as the receptor for bacterial heat-stable enterotoxin (ST) peptides and the guanylin family of gastrointestinal hormones. Activation of GC-C elevates intracellular cGMP, which modulates intestinal fluid-ion homeostasis and differentiation of enterocytes along the crypt-villus axis. GC-C activity can regulate colonic cell proliferation by inducing cell cycle arrest, and mice lacking GC-C display increased cell proliferation in colonic crypts. Activation of GC-C by administration of ST to wild type, but not Gucy2c−/−, mice resulted in a reduction in carcinogen-induced aberrant crypt foci formation. In p53-deficient human colorectal carcinoma cells, ST led to a transcriptional up-regulation of p21, the cell cycle inhibitor, via activation of the cGMP-responsive kinase PKGII and p38 MAPK. Prolonged treatment of human colonic carcinoma cells with ST led to nuclear accumulation of p21, resulting in cellular senescence and reduced tumorigenic potential. Our results, therefore, identify downstream effectors for GC-C that contribute to regulating intestinal cell proliferation. Thus, genomic responses to a bacterial toxin can influence intestinal neoplasia and senescence. PMID:24217248

  16. Albumin, in the Presence of Calcium, Elicits a Massive Increase in Extracellular Bordetella Adenylate Cyclase Toxin

    PubMed Central

    Gonyar, Laura A.; Gray, Mary C.; Christianson, Gregory J.; Mehrad, Borna

    2017-01-01

    ABSTRACT Pertussis (whooping cough), caused by Bordetella pertussis, is resurging in the United States and worldwide. Adenylate cyclase toxin (ACT) is a critical factor in establishing infection with B. pertussis and acts by specifically inhibiting the response of myeloid leukocytes to the pathogen. We report here that serum components, as discovered during growth in fetal bovine serum (FBS), elicit a robust increase in the amount of ACT, and ≥90% of this ACT is localized to the supernatant, unlike growth without FBS, in which ≥90% is associated with the bacterium. We have found that albumin, in the presence of physiological concentrations of calcium, acts specifically to enhance the amount of ACT and its localization to the supernatant. Respiratory secretions, which contain albumin, promote an increase in amount and localization of active ACT that is comparable to that elicited by serum and albumin. The response to albumin is not mediated through regulation of ACT at the transcriptional level or activation of the Bvg two-component system. As further illustration of the specificity of this phenomenon, serum collected from mice that lack albumin does not stimulate an increase in ACT. These data, demonstrating that albumin and calcium act synergistically in the host environment to increase production and release of ACT, strongly suggest that this phenomenon reflects a novel host-pathogen interaction that is central to infection with B. pertussis and other Bordetella species. PMID:28396321

  17. Biased activity of soluble guanylyl cyclase: the Janus face of thymoquinone.

    PubMed

    Detremmerie, Charlotte; Vanhoutte, Paul M; Leung, Susan

    2017-07-01

    The natural compound thymoquinone, extracted from Nigella sativa (black cumin), is widely used in humans for its anti-oxidative properties. Thymoquinone is known for its acute endothelium-independent vasodilator effects in isolated rat aortae and pulmonary arteries, depending in part on activation of adenosine triphosphate-sensitive potassium channels and inhibition of voltage-dependent calcium channels. The compound also improves endothelial dysfunction in mesenteric arteries of ageing rodents and in aortae of rabbits treated with pyrogallol, by inhibiting oxidative stress. Serendipitously, thymoquinone was found to augment contractions in isolated arteries with endothelium of both rats and pigs. The endothelium-dependent augmentation it causes counterintuitively depends on biased activation of soluble guanylyl cyclase (sGC) producing inosine 3',5'-cyclic monophosphate (cyclic IMP) rather than guanosine 3',5'-cyclic monophosphate. This phenomenon shows a striking mechanistic similarity to the hypoxic augmentation previously observed in porcine coronary arteries. The cyclic IMP preferentially produced under thymoquinone exposure causes an increased contractility of arterial smooth muscle by interfering with calcium homeostasis. This brief review summarizes the vascular pharmacology of thymoquinone, focussing in particular on how the compound causes endothelium-dependent contractions by biasing the activity of sGC.

  18. Characterisation of Two Oxidosqualene Cyclases Responsible for Triterpenoid Biosynthesis in Ilex asprella

    PubMed Central

    Zheng, Xiasheng; Luo, Xiuxiu; Ye, Guobing; Chen, Ye; Ji, Xiaoyu; Wen, Lingling; Xu, Yaping; Xu, Hui; Zhan, Ruoting; Chen, Weiwen

    2015-01-01

    Ilex asprella, a plant widely used as a folk herbal drug in southern China, produces and stores a large amount of triterpenoid saponins, most of which are of the α-amyrin type. In this study, two oxidosqualene cyclase (OSC) cDNAs, IaAS1 and IaAS2, were cloned from the I. asprella root. Functional characterisation was performed by heterologous expression in the yeast Saccharomyces cerevisiae. Analysis of the resulting products by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) showed that both genes encode a mixed amyrin synthase, producing α-amyrin and β-amyrin at different ratios. IaAS1, which mainly produces α-amyrin, is the second triterpene synthase so far identified in which the level of α-amyrin produced is ≥80% of total amyrin production. By contrast, IaAS2 mainly synthesises β-amyrin, with a yield of 95%. Gene expression patterns of these two amyrin synthases in roots and leaves of I. asprella were found to be consistent with the content patterns of total saponins. Finally, phylogenetic analysis and multiple sequence alignment of the two amyrin synthases against several known OSCs from other plants were conducted to further elucidate their evolutionary relationship. PMID:25664861

  19. Albumin, in the Presence of Calcium, Elicits a Massive Increase in Extracellular Bordetella Adenylate Cyclase Toxin.

    PubMed

    Gonyar, Laura A; Gray, Mary C; Christianson, Gregory J; Mehrad, Borna; Hewlett, Erik L

    2017-06-01

    Pertussis (whooping cough), caused by Bordetella pertussis , is resurging in the United States and worldwide. Adenylate cyclase toxin (ACT) is a critical factor in establishing infection with B. pertussis and acts by specifically inhibiting the response of myeloid leukocytes to the pathogen. We report here that serum components, as discovered during growth in fetal bovine serum (FBS), elicit a robust increase in the amount of ACT, and ≥90% of this ACT is localized to the supernatant, unlike growth without FBS, in which ≥90% is associated with the bacterium. We have found that albumin, in the presence of physiological concentrations of calcium, acts specifically to enhance the amount of ACT and its localization to the supernatant. Respiratory secretions, which contain albumin, promote an increase in amount and localization of active ACT that is comparable to that elicited by serum and albumin. The response to albumin is not mediated through regulation of ACT at the transcriptional level or activation of the Bvg two-component system. As further illustration of the specificity of this phenomenon, serum collected from mice that lack albumin does not stimulate an increase in ACT. These data, demonstrating that albumin and calcium act synergistically in the host environment to increase production and release of ACT, strongly suggest that this phenomenon reflects a novel host-pathogen interaction that is central to infection with B. pertussis and other Bordetella species. Copyright © 2017 American Society for Microbiology.

  20. Structure of RNA 3′-phosphate cyclase bound to substrate RNA

    PubMed Central

    Desai, Kevin K.; Bingman, Craig A.; Cheng, Chin L.; Phillips, George N.

    2014-01-01

    RNA 3′-phosphate cyclase (RtcA) catalyzes the ATP-dependent cyclization of a 3′-phosphate to form a 2′,3′-cyclic phosphate at RNA termini. Cyclization proceeds through RtcA–AMP and RNA(3′)pp(5′)A covalent intermediates, which are analogous to intermediates formed during catalysis by the tRNA ligase RtcB. Here we present a crystal structure of Pyrococcus horikoshii RtcA in complex with a 3′-phosphate terminated RNA and adenosine in the AMP-binding pocket. Our data reveal that RtcA recognizes substrate RNA by ensuring that the terminal 3′-phosphate makes a large contribution to RNA binding. Furthermore, the RNA 3′-phosphate is poised for in-line attack on the P–N bond that links the phosphorous atom of AMP to Nε of His307. Thus, we provide the first insights into RNA 3′-phosphate termini recognition and the mechanism of 3′-phosphate activation by an Rtc enzyme. PMID:25161314

  1. The Oxidosqualene Cyclase from the Oomycete Saprolegnia parasitica Synthesizes Lanosterol as a Single Product

    PubMed Central

    Dahlin, Paul; Srivastava, Vaibhav; Bulone, Vincent; McKee, Lauren S.

    2016-01-01

    The first committed step of sterol biosynthesis is the cyclisation of 2,3-oxidosqualene to form either lanosterol (LA) or cycloartenol (CA). This is catalyzed by an oxidosqualene cyclase (OSC). LA and CA are subsequently converted into various sterols by a series of enzyme reactions. The specificity of the OSC therefore determines the final composition of the end sterols of an organism. Despite the functional importance of OSCs, the determinants of their specificity are not well understood. In sterol-synthesizing oomycetes, recent bioinformatics, and metabolite analysis suggest that LA is produced. However, this catalytic activity has never been experimentally demonstrated. Here, we show that the OSC of the oomycete Saprolegnia parasitica, a severe pathogen of salmonid fish, has an uncommon sequence in a conserved motif important for specificity. We present phylogenetic analysis revealing that this sequence is common to sterol-synthesizing oomycetes, as well as some plants, and hypothesize as to the evolutionary origin of some microbial sequences. We also demonstrate for the first time that a recombinant form of the OSC from S. parasitica produces LA exclusively. Our data pave the way for a detailed structural characterization of the protein and the possible development of specific inhibitors of oomycete OSCs for disease control in aquaculture. PMID:27881978

  2. The Rhizobium etli cyaC Product: Characterization of a Novel Adenylate Cyclase Class

    PubMed Central

    Téllez-Sosa, Juan; Soberón, Nora; Vega-Segura, Alicia; Torres-Márquez, María E.; Cevallos, Miguel A.

    2002-01-01

    Adenylate cyclases (ACs) catalyze the formation of 3′,5′-cyclic AMP (cAMP) from ATP. A novel AC-encoding gene, cyaC, was isolated from Rhizobium etli by phenotypic complementation of an Escherichia coli cya mutant. The functionality of the cyaC gene was corroborated by its ability to restore cAMP accumulation in an E. coli cya mutant. Further, overexpression of a malE::cyaC fusion protein allowed the detection of significant AC activity levels in cell extracts of an E. coli cya mutant. CyaC is unrelated to any known AC or to any other protein exhibiting a currently known function. Thus, CyaC represents the first member of a novel class of ACs (class VI). Hypothetical genes of unknown function similar to cyaC have been identified in the genomes of the related bacterial species Mesorhizobium loti, Sinorhizobium meliloti, and Agrobacterium tumefaciens. The cyaC gene is cotranscribed with a gene similar to ohr of Xanthomonas campestris and is expressed only in the presence of organic hydroperoxides. The physiological performance of an R. etli cyaC mutant was indistinguishable from that of the wild-type parent strain both under free-living conditions and during symbiosis. PMID:12057950

  3. Effects of forskolin on cerebral blood flow: implications for a role of adenylate cyclase

    SciTech Connect

    Wysham, D.G.; Brotherton, A.F.; Heistad, D.D.

    1986-11-01

    We have studied cerebral vascular effects of forskolin, a drug which stimulates adenylate cyclase and potentiates dilator effects of adenosine in other vascular beds. Our goals were to determine whether forskolin is a cerebral vasodilator and whether it potentiates cerebral vasodilator responses to adenosine. We measured cerebral blood flow with microspheres in anesthetized rabbits. Forskolin (10 micrograms/kg per min) increased blood flow (ml/min per 100 gm) from 39 +/- 5 (mean +/- S.E.) to 56 +/- 9 (p less than 0.05) in cerebrum, and increased flow to myocardium and kidney despite a decrease in mean arterial pressure. Forskolin did notmore » alter cerebral oxygen consumption, which indicates that the increase in cerebral blood flow is a direct vasodilator effect and is not secondary to increased metabolism. We also examined effects of forskolin on the response to infusion of adenosine. Cerebral blood flow was measured during infusion of 1-5 microM/min adenosine into one internal carotid artery, under control conditions and during infusion of forskolin at 3 micrograms/kg per min i.v. Adenosine alone increased ipsilateral cerebral blood flow from 32 +/- 3 to 45 +/- 5 (p less than 0.05). Responses to adenosine were not augmented during infusion of forskolin. We conclude that forskolin is a direct cerebral vasodilator and forskolin does not potentiate cerebral vasodilator responses to adenosine.« less

  4. Guanylyl cyclase-dependent chemotaxis of endothelial cells in response to nitric oxide gradients.

    PubMed

    Isenberg, Jeff S; Ridnour, Lisa A; Thomas, Douglas D; Wink, David A; Roberts, David D; Espey, Michael Graham

    2006-03-15

    Nitric oxide (NO) is an important regulator of angiogenesis and neovascularization. The nature of endothelial cell motility responses to NO was examined using a Boyden chamber method. NO generated via decomposition of either DEA/NO or DETA/NO produced increases in human umbilical vein endothelial cell (HUVEC) chemotaxis, which were completely abrogated by ODQ, a soluble guanylyl cyclase inhibitor. Measurements of NO either directly by chemiluminescence or its chemistry with diaminofluorescein revealed that chemotaxis was driven by subtle NO gradients between the lower and the upper wells in this system. In addition to diffusion and volatilization from the upper chambers, the data showed that HUVEC consumption of NO contributed to these sustained gradients. Comparison of DEA/NO- and DETA/NO-mediated responses suggested that the persistence of spatial NO gradients is as significant as the absolute magnitude of NO exposure per unit time. The findings suggest that subnanomolar NO gradients are sufficient to mobilize endothelial cell migration into hypoxic tissue during neovascularization events, such as in wound healing and cancer.

  5. Natriuretic Peptide Receptor Guanylyl Cyclase-A Protects Podocytes from Aldosterone-Induced Glomerular Injury

    PubMed Central

    Ogawa, Yoshihisa; Yokoi, Hideki; Kasahara, Masato; Mori, Kiyoshi; Kato, Yukiko; Kuwabara, Takashige; Imamaki, Hirotaka; Kawanishi, Tomoko; Koga, Kenichi; Ishii, Akira; Tokudome, Takeshi; Kishimoto, Ichiro; Sugawara, Akira; Nakao, Kazuwa

    2012-01-01

    Natriuretic peptides produced by the heart in response to cardiac overload exert cardioprotective and renoprotective effects by eliciting natriuresis, reducing BP, and inhibiting cell proliferation and fibrosis. These peptides also antagonize the renin-angiotensin-aldosterone system, but whether this mechanism contributes to their renoprotective effect is unknown. Here, we examined the kidneys of mice lacking the guanylyl cyclase-A (GC-A) receptor for natriuretic peptides under conditions of high aldosterone and high dietary salt. After 4 weeks of administering aldosterone and a high-salt diet, GC-A knockout mice, but not wild-type mice, exhibited accelerated hypertension with massive proteinuria. Aldosterone-infused GC-A knockout mice had marked mesangial expansion, segmental sclerosis, severe podocyte injury, and increased oxidative stress. Reducing the BP with hydralazine failed to lessen such changes; in contrast, blockade of the renin-angiotensin-aldosterone system markedly reduced albuminuria, ameliorated podocyte injury, and reduced oxidative stress. Furthermore, treatment with the antioxidant tempol significantly reduced albuminuria and abrogated the histologic changes. In cultured podocytes, natriuretic peptides inhibited aldosterone-induced mitogen-activated protein kinase phosphorylation. Taken together, these results suggest that renoprotective properties of the endogenous natriuretic peptide/GC-A system may result from the local inhibition of the renin-angiotensin-aldosterone system and oxidative stress in podocytes. PMID:22652704

  6. Inhibitors for human glutaminyl cyclase by structure based design and bioisosteric replacement.

    PubMed

    Buchholz, Mirko; Hamann, Antje; Aust, Susanne; Brandt, Wolfgang; Böhme, Livia; Hoffmann, Torsten; Schilling, Stephan; Demuth, Hans-Ulrich; Heiser, Ulrich

    2009-11-26

    The inhibition of human glutaminyl cyclase (hQC) has come into focus as a new potential approach for the treatment of Alzheimer's disease. The hallmark of this principle is the prevention of the formation of Abeta(3,11(pE)-40,42), as these Abeta-species were shown to be of elevated neurotoxicity and likely to act as a seeding core leading to an accelerated formation of Abeta-oligomers and fibrils. Starting from 1-(3-(1H-imidazol-1-yl)propyl)-3-(3,4-dimethoxyphenyl)thiourea, bioisosteric replacements led to the development of new classes of inhibitors. The optimization of the metal-binding group was achieved by homology modeling and afforded a first insight into the probable binding mode of the inhibitors in the hQC active site. The efficacy assessment of the hQC inhibitors was performed in cell culture, directly monitoring the inhibition of Abeta(3,11(pE)-40,42) formation.

  7. Enzymatic Addition of Alcohols to Terpenes by Squalene Hopene Cyclase Variants.

    PubMed

    Kühnel, Lisa C; Nestl, Bettina M; Hauer, Bernhard

    2017-11-16

    Squalene-hopene cyclases<