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Cyclase
Kinds of Cyclase Terms modified by Cyclase Selected AbstractsEffects of Straight Chain Alcohols on Specific Isoforms of Adenylyl CyclaseALCOHOLISM, Issue 4 2010Mohammad Hasanuzzaman Background:, Our previous studies showed that the activity of adenylyl cyclase (AC) was enhanced by pharmacologically relevant concentrations of ethanol, that this enhancing effect of ethanol on AC activity was AC isoform specific, and that the alcohol cutoff effect for n -alkanol potentiation of AC activity was also AC isoform specific. Therefore, we hypothesized that within the cyclic AMP-generating system, AC is the target of ethanol's action and that alcohols interact directly with the AC molecules. To characterize the interaction between alcohols and AC proteins, the effects of a series of straight chain alcohols would be very valuable in understanding alcohol action at the molecular level. To our knowledge, straight chain alcohols other than n- alkanols and 1,,-diols have not been used extensively to study alcohol effects on the activity of AC or other proteins important in the alcohol research field. Methods:, The effects of a series of straight chain alcohols on D1A dopamine receptor-stimulated activity of AC isoforms type 6, 7, and 9 (AC6, AC7, and AC9) were examined in transfected Hela cells by a cAMP accumulation assay. Results:, In general, all 3 AC isoforms responded to a series of straight chain alcohols in a similar manner. The order of responsiveness is as follows: monoalcohol > diol > triol and tetraol. Within monoalcohols, 1-alcohols had larger effects than 2-alcohols. Two of 3 stereoisomers of 2,3-butanediol, [D-(-)-2,3-butanediol and meso -2,3-butanediol] showed similar enhancing effects on all 3 AC isoforms. However, the third stereoisomer, L-(+)-2,3-butanediol, inhibited AC7 activity, while it stimulated AC6 and AC9. Conclusion:, The number and the position of hydroxyl groups in straight chain alcohols play an important role in the magnitude of the enhancement on AC activity. Regardless of AC isoforms, the most effective of the straight chain alcohols seems to be the 1-alcohol (n -alkanol) for a given chain length. We found that one of the stereoisomers of 2,3-butanediol had opposite effects on AC activity depending on the AC isoform. Overall, the results are consistent with the hypotheses and demonstrate that a series of straight chain alcohols can be a valuable tool to study AC-alcohol interactions. [source] Alcohol Stimulates Ciliary Motility of Isolated Airway Axonemes Through a Nitric Oxide, Cyclase, and Cyclic Nucleotide-Dependent Kinase MechanismALCOHOLISM, Issue 4 2009Joseph H. Sisson Background:, Lung mucociliary clearance provides the first line of defense from lung infections and is impaired in individuals who consume heavy amounts of alcohol. Previous studies have demonstrated that this alcohol-induced ciliary dysfunction occurs through impairment of nitric oxide (NO) and cyclic nucleotide-dependent kinase-signaling pathways in lung airway ciliated epithelial cells. Recent studies have established that all key elements of this alcohol-driven signaling pathway co-localize to the apical surface of the ciliated cells with the basal bodies. These findings led us to hypothesize that alcohol activates the cilia stimulation pathway at the organelle level. To test this hypothesis we performed experiments exposing isolated demembranated cilia (isolated axonemes) to alcohol and studied the effect of alcohol-stimulated ciliary motility on the pathways involved with isolated axoneme activation. Methods:, Isolated demembranated cilia were prepared from bovine trachea and activated with adenosine triphosphate. Ciliary beat frequency, NO production, adenylyl and guanylyl cyclase activities, cAMP- and cGMP-dependent kinase activities were measured following exposure to biologically relevant concentrations of alcohol. Results:, Alcohol rapidly stimulated axoneme beating 40% above baseline at very low concentrations of alcohol (1 to 10 mM). This activation was specific to ethanol, required the synthesis of NO, the activation of soluble adenylyl cyclase (sAC), and the activation of both cAMP- and cGMP-dependent kinases (PKA and PKG), all of which were present in the isolated organelle preparation. Conclusions:, Alcohol rapidly and sequentially activates the eNOS,NO,GC,cGMP,PKG and sAC,cAMP, PKA dual signaling pathways in isolated airway axonemes. These findings indicate a direct effect of alcohol on airway cilia organelle function and fully recapitulate the alcohol-driven activation of cilia known to exist in vivo and in intact lung ciliated cells in vitro following brief moderate alcohol exposure. Furthermore, these findings indicate that airway cilia are exquisitely sensitive to the effects of alcohol and substantiate a key role for alcohol in the alterations of mucociliary clearance associated with even low levels of alcohol intake. We speculate that this same axoneme-based alcohol activation pathway is down regulated following long-term high alcohol exposure and that the isolated axoneme preparation provides an excellent model for studying the mechanism of alcohol-mediated cilia dysfunction. [source] Enzymatic Formation of Multiple Triterpenes by Mutation of Tyrosine 510 of the Oxidosqualene-Lanosterol Cyclase from Saccharomyces cerevisiaeCHEMBIOCHEM, Issue 12 2004Tung-Kung Wu Prof. Dr. The simultaneous formation of monocyclic and differentially deprotonated triterpenes by mutation of Tyr510 of the oxidosqualene-lanosterol cyclase (ERG7) from Saccharomyces cerevisiae is reported (see scheme). Possible dual functions of this residue involved in the cyclization and deprotonation steps of the oxidosqualene cyclization/rearrangement cascade are discussed. [source] Synthesis of (E)- and (Z)-29-Methylidyne-2,3-oxidosqualene Derivatives as Inhibitors of Liver and Yeast Oxidosqualene Cyclase.CHEMINFORM, Issue 47 2002Maurizio Ceruti Abstract For Abstract see ChemInform Abstract in Full Text. [source] A novel role of differentiation-inducing factor-1 in Dictyostelium development, assessed by the restoration of a developmental defect in a mutant lacking mitogen-activated protein kinase ERK2DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5 2000Hidekazu Kuwayama It has been previously reported that the differentiating wild-type cells of Dictyostelium discoideum secrete a diffusible factor or factors that are able to rescue the developmental defect in the mutant lacking extracellular signal-regulated kinase 2 (ERK2), encoded by the gene erkB. In the present study, it is demonstrated that differentiation-inducing factor-1 (DIF-1) for stalk cells can mimic the role of the factor(s) and the mechanism of the action of DIF-1 in the erkB null mutant is also discussed. The mutant usually never forms multicellular aggregates, because of its defect in cyclic adenosine monophosphate (cAMP) signaling. In the presence of 100 n M DIF-1, however, the mutant cells formed tiny slugs, which eventually developed into small fruiting bodies. In contrast, DIF-1 never rescued the developmental arrest of other Dictyostelium mutants lacking adenylyl cyclase A (ACA), cAMP receptors cAR1 and cAR3, heterotrimeric G-protein, the cytosolic regulator of ACA, or the catalytic subunit of cAMP-dependent protein kinase (PKA-C). Most importantly, it was found that DIF-1 did not affect the cellular cAMP level, but rather elevated the transcriptional level of pka during the development of erkB null cells. These results suggest that DIF-1 may rescue the developmental defect in erkB null cells via the increase in PKA activity, thus giving the first conclusive evidence that DIF-1 plays a crucial role in the early events of Dictyostelium development as well as in prestalk and stalk cell induction. [source] G,, that interacts with adenylyl cyclase in opioid tolerance originates from a Gs proteinDEVELOPMENTAL NEUROBIOLOGY, Issue 12 2006Hoau-Yan Wang Abstract We previously demonstrated that chronic morphine induces a change in G protein coupling by the mu opioid receptor (MOR) from Gi/o to Gs, concurrent with the instatement of an interaction between G,, and adenylyl cyclase types II and IV. These two signaling changes confer excitatory effects on the cell in place of the typical inhibition by opioids and are associated with morphine tolerance and dependence. Both signaling changes and these behavioral manifestations of chronic morphine are attenuated by cotreatment with ultra-low-dose naloxone. In the present work, using striatum from chronic morphine-treated rats, we isotyped the G, within Gs and Go heterotrimers that coupled to MOR and compared these to the G, isotype of the G,, that interacted with adenylyl cyclase II or IV after chronic morphine treatment. Isotyping results show that chronic morphine causes a Gs heterotrimer associated with MOR to release its G,, to interact with adenylyl cyclase. These data suggest that the switch to Gs coupling by MOR in response to chronic morphine, which is attenuated by ultra-low-dose opioid antagonist cotreatment, leads to a two-pronged stimulation of adenylyl cyclase utilizing both G, and G,, subunits of the Gs protein novel to this receptor. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006 [source] Activation of receptors negatively coupled to adenylate cyclase is required for induction of long-term synaptic depression at Schaffer collateral-CA1 synapsesDEVELOPMENTAL NEUROBIOLOGY, Issue 3 2006Linda A. Santschi Abstract Chemical LTD (CLTD) of synaptic transmission is triggered by simultaneously increasing presynaptic [cGMP] while inhibiting PKA. Here, we supply evidence that class II, but not III, metabotropic glutamate receptors (mGluRs), and A1 adenosine receptors, both negatively coupled to adenylate cyclase, play physiologic roles in providing PKA inhibition necessary to promote the induction of LTD at Schaffer collateral-CA1 synapses in hippocampal slices. Simultaneous activation of group II mGluRs with the selective agonist (2S,2,R,3,R)-2-(2,,3,-dicarboxy-cyclopropyl) glycine (DCGIV; 5 ,M), while raising [cGMP] with the type V phosphodiesterase inhibitor, zaprinast (20 ,M), resulted in a long-lasting depression of synaptic strength. When zaprinast (20 ,M) was combined with a cell-permeant PKA inhibitor H-89 (10 ,M), the need for mGluR IIs was bypassed. DCGIV, when combined with a "submaximal" low frequency stimulation (1 Hz/400 s), produced a saturating LTD. The mGluR II selective antagonist, (2S)-alpha-ethylglutamic acid (EGLU; 5 ,M), blocked induction of LTD by prolonged low frequency stimulation (1 Hz/900 s). In contrast, the mGluR III selective receptor blocker, (RS)-a-Cyclopropyl-[3- 3H]-4-phosphonophenylglycine (CPPG; 10 ,M), did not impair LTD. The selective adenosine A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 100 nM), also blocked induction of LTD, while the adenosine A1 receptor agonist N6 -cyclohexyl adenosine (CHA; 50 nM) significantly enhanced the magnitude of LTD induced by submaximal LFS and, when paired with zaprinast (20 ,M), was sufficient to elicit CLTD. Inhibition of PKA with H-89 rescued the expression of LTD in the presence of either EGLU or DPCPX, confirming the hypothesis that both group II mGluRs and A1 adenosine receptors enhance the induction of LTD by inhibiting adenylate cyclase and reducing PKA activity. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2006 [source] Xanthine-analog, KMUP-2, enhances cyclic GMP and K+ channel activities in rabbit aorta and corpus cavernosum with associated penile erectionDRUG DEVELOPMENT RESEARCH, Issue 3 2002Rong-Jyh Lin Abstract The pharmacological properties of KMUP-2 were examined in isolated rabbit aorta and corpus cavernosum smooth muscle (CCSM). KMUP-2 caused relaxations that were attenuated by removed endothelium, high K+, and pretreatment with the soluble guanylate cyclase (sGC) inhibitors methylene blue (10 ,M) and ODQ (1 ,M), a NOS inhibitor, L-NAME (100 ,M), a K+ channel blocker TEA (10 mM), a KATP channel blocker glibenclamide (1 ,M), a voltage-dependent K+ channel blocker 4-AP (100 ,M), and the Ca2+ -dependent K+ channel blockers apamin (1 ,M) and charybdotoxin (ChTX, 0.1 ,M). The relaxant responses of KMUP-2 (0.01, 0.05, 0.1 ,M) together with a PDE inhibitor, IBMX (0.5 ,M), had additive effects on rabbit aorta and CCSM. Additionally, KMUP-2 (100 ,M) also affected cGMP metabolism, due to its inhibiting activity on PDE in human platelets. KMUP-2 (0.1,100 ,M) further induced an increase of intracellular cGMP levels in the primary cultured rabbit aortic and CCSM cells. These increases in cGMP content were abolished in the presence of methylene blue (100 ,M) and ODQ (10 ,M). Obviously, the relaxant effects of KMUP-2 on rabbit isolated tissues are more sensitive in CCSM than in aorta. Moreover, KMUP-2 also stimulated NO/sGC/cGMP pathway and subsequent elevation of cGMP by blockade of PDE and enhanced opening of K+ channels in rabbit aorta and CCSM. KMUP-2 (0.2, 0.4, 0.6 mg/kg), similar to KMUP-1 and sildenafil, caused increases of intracavernous pressure (ICP) and duration of tumescene (DT) in a dose-dependent manner. It is concluded that both the increases of cGMP and the opening activity of K+ channels play prominent roles in KMUP-2-induced aortic smooth muscle and CCSM relaxation and increases of ICP in rabbits. Drug Dev. Res. 55:162,172, 2002. © 2002 Wiley-Liss, Inc. [source] Discovery and recognition of purine receptor subtypes on plateletsDRUG DEVELOPMENT RESEARCH, Issue 1-2 2001Susanna M.O. HouraniArticle first published online: 9 MAY 200 Abstract The effects of purines on platelets have been known since the 1960s, when Born demonstrated aggregation induced by ADP and its inhibition by adenosine and by ATP. The inhibition by adenosine is not specific for ADP, and adenosine acts at a separate receptor to stimulate adenylate cyclase, which has an inhibitory effect on platelet function. Studies using selective agonists and antagonists have shown that the platelet receptor is of the A2A subtype and this has been confirmed using A2A knockout mice. The situation with ADP is more complex, and there has been controversy about the number of ADP receptors on platelets. ADP causes shape change, aggregation, mobilisation of calcium from intracellular stores, rapid calcium influx, and inhibition of adenylate cyclase, and the relationship between these is becoming clearer. Two cloned P2 receptors have been detected on platelets, P2X1 and P2Y1, and a third P2Y receptor is thought to exist. The P2X1 receptor is responsible for the rapid calcium influx and can be activated by ATP as well as by ADP, but is likely to be desensitised under normal experimental conditions and its pathophysiological role is uncertain. The P2Y1 receptor is responsible for calcium mobilisation, shape change, and the initiation of aggregation, and these responses are abolished in P2Y1 knockout mice, while the other P2Y receptor is responsible for inhibition of adenylate cyclase and is required for full aggregation. ATP is a competitive antagonist at both these P2Y receptors, while some nucleotide analogues can discriminate between them. Drug Dev. Res. 52:140,149, 2001. © 2001 Wiley-Liss, Inc. [source] Atrazine increases the sodium absorption in frog (Rana esculenta) skinENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 2 2006Giuseppe Cassano Abstract The presence of atrazine in agricultural sites has been linked to the decline in amphibian populations. The efforts of the scientific community generally are directed toward investigating the long-term effect of atrazine on complex functions (reproduction or respiration), but in the present study, we investigated the short-term effect on the short-circuit current (ISC), a quantitative measure of the ion transport operated by frog (Rana esculenta) skin. Treatment with 5 ,M atrazine (1.08 mg/L) does not affect the transepithelial outfluxes of [14C]mannitol or [14C]urea; therefore, atrazine does not damage the barrier properties of frog skin. Atrazine causes a dose-dependent increase in the short-circuit current, with a minimum of 4.64 ± 0.76 ,A/cm2 (11.05% ± 1.22%) and a maximum of 12.7 ± 0.7 ,A/cm2 (35% ± 2.4%) measured at 10 nM and 5 ,M, respectively. An increase in ISC also is caused by 5 ,M ametryne, prometryn, simazine, terbuthylazine, or terbutryn (other atrazine derivatives). In particular, atrazine increases the transepithelial 22Na+ influx without affecting the outflux. Finally, stimulation of ISC by atrazine is suppressed by SQ 22536, H89, U73122, 2-aminoethoxydiphenyl borate, and W7 (blockers of adenylate cyclase, protein kinase A, phospholipase C, intracellular Ca2+ increase, and calmodulin, respectively), whereas indomethacin and calphostin C (inhibitors of cyclooxygenase and protein kinase C, respectively) have no effect. [source] Adenylyl cyclase encoded by AC78C participates in sugar perception in Drosophila melanogasterEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2008Kohei Ueno Abstract In gustatory receptor neurons (GRNs) in Drosophila melanogaster, Gr5a and one of the Gr64s encode sugar receptors with seven transmembrane domains. Previously, we have shown that the responses to various sugars are depressed in DGs, mutant flies (Ueno et al., 2006). Because DGs, is a homolog of Gs, we hypothesized that the sugar receptors are coupled to adenylyl cyclase (AC) in Drosophila. The aim of this study was to identify the AC that participates in sugar perception. Here, we found that an AC inhibitor, MDL-12330A, depressed the response in GRNs to trehalose as well as sucrose; that an AC gene, AC78C, was expressed in the sugar-sensitive GRNs; that RNAi against AC78C depressed the electrical response in GRNs to sucrose; and that the sugar response in GRNs, as well as sugar intake in a behavioral assay in an AC78C mutant, was depressed at low sugar concentrations. We conclude that AC78C, via cAMP, participates in the sugar-taste signaling pathway at the low concentration range. [source] The nitric oxide/cyclic guanosine monophosphate pathway modulates the inspiratory-related activity of hypoglossal motoneurons in the adult ratEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2008Fernando Montero Abstract Motoneurons integrate interneuronal activity into commands for skeletal muscle contraction and relaxation to perform motor actions. Hypoglossal motoneurons (HMNs) are involved in essential motor functions such as breathing, mastication, swallowing and phonation. We have investigated the role of the gaseous molecule nitric oxide (NO) in the regulation of the inspiratory-related activity of HMNs in order to further understand how neural activity is transformed into motor activity. In adult rats, we observed nitrergic fibers and bouton-like structures in close proximity to motoneurons, which normally lack the molecular machinery to synthesize NO. In addition, immunohistochemistry studies demonstrated that perfusion of animals with a NO donor resulted in an increase in the levels of cyclic guanosine monophosphate (cGMP) in motoneurons, which express the soluble guanylyl cyclase (sGC) in the hypoglossal nucleus. Modulators of the NO/cGMP pathway were micro-iontophoretically applied while performing single-unit extracellular recordings in the adult decerebrated rat. Application of a NO synthase inhibitor or a sGC inhibitor induced a statistically significant reduction in the inspiratory-related activity of HMNs. However, excitatory effects were observed by ejection of a NO donor or a cell-permeable analogue of cGMP. In slice preparations, application to the bath of a NO donor evoked membrane depolarization and a decrease in rheobase, which were prevented by co-addition to the bath of a sGC inhibitor. These effects were not prevented by reduction of the spontaneous synaptic activity. We conclude that NO from afferent fibers anterogradely modulates the inspiratory-related activity of HMNs by a cGMP-dependent mechanism in physiological conditions. [source] Peripheral antinociceptive effect of pertussis toxin: activation of the arginine/NO/cGMP/PKG/ ATP-sensitive K+ channel pathwayEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2006Gerly A. C. Brito Abstract The aim of the present study was to determine the effect of pertussis toxin (PTX) on inflammatory hypernociception measured by the rat paw pressure test and to elucidate the mechanism involved in this effect. In this test, prostaglandin E2 (PGE2) administered subcutaneously induces hypernociception via a mechanism associated with neuronal cAMP increase. Local intraplantar pre-treatment (30 min before), and post-treatment (5 min after) with PTX (600 ng/paw1, in 100 µL) reduced hypernociception induced by prostaglandin E2 (100 ng/paw, in 100 µL, intraplantar). Furthermore, local intraplantar pre-treatment (30 min before) with PTX (600 ng/paw, in 100 µL) reduced hypernociception induced by DbcAMP, a stable analogue of cAMP (100 µg/paw, in 100 µL, intraplantar), which indicates that PTX may have an effect other than just Gi/G0 inhibition. PTX-induced analgesia was blocked by selective inhibitors of nitric oxide synthase (L-NMMA), guanylyl cyclase (ODQ), protein kinase G (KT5823) and ATP-sensitive K+ channel (Kir6) blockers (glybenclamide and tolbutamide). In addition, PTX was shown to induce nitric oxide (NO) production in cultured neurons of the dorsal root ganglia. In conclusion, this study shows a peripheral antinociceptive effect of pertussis toxin, resulting from the activation of the arginine/NO/cGMP/PKG/ATP-sensitive K+ channel pathway. [source] Melatonin inhibits hippocampal long-term potentiationEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2005Louisa M. Wang Abstract The goal of this study is to investigate the effect of the hormone melatonin on long-term potentiation and excitability measured by stimulating the Schaffer collaterals and recording the field excitatory postsynaptic potential from the CA1 dendritic layer in hippocampal brain slices from mice. Application of melatonin produced a concentration-dependent inhibition of the induction of long-term potentiation, with a concentration of 100 nm producing an ,,50% inhibition of long-term potentiation magnitude. Long-duration melatonin treatments of 6 h were also effective at reducing the magnitude of long-term potentiation. Melatonin (100 nm) did not alter baseline evoked responses or paired-pulse facilitation recorded at this synapse. The inhibitory actions of melatonin were prevented by application of the melatonin (MT) receptor antagonist luzindole as well as the MT2 receptor subtype antagonist 4-phenyl-2-propionamidotetraline. These inhibitory actions of melatonin were lost in mice deficient in MT2 receptors but not those deficient in MT1 receptors. In addition, application of the protein kinase A inhibitor H-89 both mimicked the effects of melatonin and precluded further inhibition by melatonin. Finally, the application an activator of adenylyl cyclase, forskolin, overcame the inhibitory effects of melatonin on LTP without affecting the induction of long-term potentiation on its own. These results suggest that hippocampal synaptic plasticity may be constrained by melatonin through a mechanism involving MT2-receptor-mediated regulation of the adenylyl cyclase,protein kinase A pathway. [source] Enhancement of learning behaviour by a potent nitric oxide-guanylate cyclase activator YC-1EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2005Wei-Lin Chien Abstract Memory is one of the most fundamental mental processes, and various approaches have been used to understand the mechanisms underlying this process. Nitric oxide (NO), cGMP and protein kinase G (PKG) are involved in the modulation of synaptic plasticity in various brain regions. YC-1, which is a benzylindazole derivative, greatly potentiated the response of soluble guanylate cyclase to NO (up to several hundreds fold). We have previously shown that YC-1 markedly enhances long-term potentiation in hippocampal and amygdala slices via NO-cGMP-PKG-dependent pathway. We here further investigated whether YC-1 promotes learning behaviour in Morris water maze and avoidance tests. It was found that YC-1 shortened the escape latency in the task of water maze, increased and decreased the retention scores in passive and active avoidance task, respectively. Administration of YC-1 30 min after foot-shock stimulation did not significantly affect retention scores in response to passive avoidance test. Administration of scopolamine, a muscarinic antagonist, markedly impaired the memory acquisition. Pretreatment of YC-1 inhibited the scopolamine-induced learning deficit. The enhancement of learning behaviour by YC-1 was antagonized by intracerebroventricular injection of NOS inhibitor L-NAME and PKG inhibitors of KT5823 and Rp-8-Br-PET-cGMPS, indicating that NO-cGMP-PKG pathway is also involved in the learning enhancement action of YC-1. YC-1 is thus a good drug candidate for the improvement of learning and memory. [source] Soluble guanylyl cyclase appears in a specific subset of periglomerular cells in the olfactory bulbEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2005Maria Gutièrrez-Mecinas Abstract In the brain, nitric oxide acts as an atypical messenger in cellular nonsynaptic transmission. In the olfactory bulb, this gas is produced at the level of the olfactory glomeruli by a subpopulation of periglomerular cells that participates in the first synaptic relay of the olfactory information between the olfactory nerve and the dendritic tufts of principal cells. It has been proposed that nitric oxide modulates intraglomerular synaptic integration of sensory inputs, but its specific role in the glomerular circuitry remains to be understood. In this article, we demonstrate that, in the glomerular circuits, a specific subset of periglomerular cells, most of them expressing the calcium binding protein calbindin D-28 k, expresses the ,1 subunit of the soluble guanylyl cyclase. These cells could be the targets for the action of nitric oxide at the glomerular level via activation of soluble guanylyl cyclase and production of cGMP. [source] Potentiation of glycine responses by dideoxyforskolin and tamoxifen in rat spinal neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2003Dominique Chesnoy-Marchais Abstract Dideoxyforskolin, a forskolin analogue unable to stimulate adenylate cyclase, and tamoxifen, an antioestrogen widely used against breast cancer, are both known to block some Cl, channels. Their effects on Cl, responses to glycine or GABA have been tested here by using whole-cell recording from cultured spinal neurons. Dideoxyforskolin (4 or 16 µm) and tamoxifen (0.2,5 µm) both potentiate responses to low glycine concentrations. They also induce blocking effects, predominant at high glycine concentrations. At 5 µm, tamoxifen increased responses to 15 µm glycine by a factor >4.5, reaching 20 in some neurons. Potentiation by extracellular dideoxyforskolin or tamoxifen persisted after intracellular application of the modulator and was not due to Zn2+ contamination. Potentiation by tamoxifen also persisted in a Ca2+ -free extracellular solution, after intracellular Ca2+ buffering and protein kinase C blockade. Thus, the critical sites of action are not intracellular. The EC50 for glycine was lowered 6.6-fold by 5 µm tamoxifen. The kinetics and voltage-dependence of the effects of tamoxifen on glycine responses support the idea that this hydrophobic drug may act from a site located within the membrane. Tamoxifen (5 µm) also increased responses to 2 µm GABA by a factor of 3.5, but barely affected peak responses to 20 µm GABA. The demonstration that tamoxifen affects some of the main inhibitory receptors should be useful for better evaluating its neurological effects. Furthermore, the results identify a new class of molecules that potentiate glycine receptor function. [source] Differential galanin receptor-1 and galanin expression by 5-HT neurons in dorsal raphé nucleus of rat and mouse: evidence for species-dependent modulation of serotonin transmissionEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2003Jari A. Larm Abstract Galanin and galanin receptors are widely expressed by neurons in rat brain that either synthesize/release and/or are responsive to, classical transmitters such as ,-aminobutyric acid, acetylcholine, noradrenaline, histamine, dopamine and serotonin (5-hydroxytryptamine, 5-HT). The dorsal raphé nucleus (DRN) contains , 50% of the 5-HT neurons in the rat brain and a high percentage of these cells coexpress galanin and are responsive to exogenous galanin in vitro. However, the precise identity of the galanin receptor(s) present on these 5-HT neurons has not been previously established. Thus, the current study used a polyclonal antibody for the galanin receptor-1 (GalR1) to examine the possible expression of this receptor within the DRN of the rat and for comparative purposes also in the mouse. In the rat, intense GalR1-immunoreactivity (IR) was detected in a substantial population of 5-HT-immunoreactive neurons in the DRN, with prominent receptor immunostaining associated with soma and proximal dendrites. GalR1-IR was also observed in many cells within the adjacent median raphé nucleus. In mouse DRN, neurons exhibited similar levels and distribution of 5-HT-IR to that in the rat, but GalR1-IR was undetectable. Consistent with this, galanin and GalR1 mRNA were also undetectable in mouse DRN by in situ hybridization histochemistry, despite the detection of GalR1 mRNA (and GalR1-IR) in adjacent cells in the periaqueductal grey and other midbrain areas. 5-HT neuron activity in the DRN is primarily regulated via 5-HT1A autoreceptors, via inhibition of adenylate cyclase and activation of inward-rectifying K+ channels. Notably, the GalR1 receptor subtype signals via identical mechanisms and our findings establish that galanin modulates 5-HT neuron activity in the DRN of the rat via GalR1 (auto)receptors. However, these studies also identify important species differences in the relationship between midbrain galanin and 5-HT systems, which should prompt further investigations in relation to comparative human neurochemistry and which have implications for studies of animal models of relevant neurological conditions such as stress, anxiety and depression. [source] Characterization of the mouse adenylyl cyclase type VIII gene promoter: regulation by cAMP and CREBEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2002Jennifer R. Chao Abstract Adenylyl cyclase (AC) type VIII has been implicated in several forms of neural plasticity, including drug addiction and learning and memory. In the present study, we directly examined the role for the transcription factor CREB (cAMP response element binding protein) in regulating ACVIII expression by cloning a 5.2 kilobase region upstream of the translation start site of the mouse ACVIII gene. Analysis of this fragment revealed consensus elements for several transcription factors, including a canonical cAMP response element (CRE) in close proximity to the transcription initiation region. Next, ACVIII promoter activity was studied in two neural-derived cell lines and in primary cultures of rat striatal neurons. Activation of the cAMP pathway by forskolin treatment increased promoter activity, and a series of deletion and point mutants demonstrated that this activation is mediated specifically via the canonical CRE site. Gel shift assays confirmed that this site can bind CREB and several CREB family proteins. Further, activation of the ACVIII promoter by forskolin was potentiated by expression of a constitutively active form of CREB, CREB-VP16, whereas it was inhibited by expression of a dominant-negative form of CREB, A-CREB. Finally, over-expression of CREB in vivo, by viral-mediated gene transfer, induced ACVIII promoter activity in the brains of ACVIII-LacZ transgenic mice. These results suggest that the ACVIII gene is regulated by CREB in vitro and in vivo and that this regulation may contribute to CREB-dependent neural plasticity. [source] Phosphorylation of voltage-gated ion channels in rat olfactory receptor neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2001Christian H. Wetzel Abstract In olfactory receptor neurons (ORNs), ligand,odorant receptor interactions cause G protein-mediated activation of adenylate cyclase and a subsequent increase in concentration of the intracellular messenger cAMP. Odorant-evoked elevation in cAMP is thought to directly activate a cation-selective cyclic nucleotide-gated channel, which causes external Ca2+ influx, leading to membrane depolarization and the generation of action potentials. Our data show that in freshly dissociated rat ORNs, odorant-induced elevation in cAMP also activates cAMP-dependent protein kinase (PKA), which is then able to phosphorylate various protein targets in the olfactory signal transduction pathway, specifically voltage-gated sodium and calcium channels. The presence of PKI (PKA inhibitor peptide) blocked the modulatory action of cAMP on voltage-gated ion channels. By modulating the input/output properties of the sensory neurons, this mechanism could take part in the complex adaptation process in odorant perception. In addition, we found modulation of voltage-gated sodium and calcium channel currents by 5-hydroxytryptamine and the dopamine D1 receptor agonist SKF 38393. These findings suggest that in situ ORNs might also be a target for efferent modulation. [source] Exogenous nitric oxide causes potentiation of hippocampal synaptic transmission during low-frequency stimulation via the endogenous nitric oxide,cGMP pathwayEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2001Christelle L. M. Bon Abstract Nitric oxide (NO) is a putative participant in synaptic plasticity and demonstrations that exogenous NO can elicit the same plastic changes have been taken to support such a role. The experiments, carried out on the CA1 region of rat hippocampal slices, were aimed at testing this interpretation. A major component of tetanus-induced long-term potentiation (LTP) was lost in response to l -nitroarginine, which inhibits NO synthase, and 1H -[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (ODQ), which inhibits NO-sensitive soluble guanylyl cyclase (sGC). At 0.2 Hz afferent fibre stimulation, exogenous NO produced, concentration-dependently, a synaptic depression that reverted on washout to a persistent potentiation that occluded tetanus-induced LTP. The NO concentrations necessary (estimated in the 100-nm range), however, were mostly supramaximal for stimulating hippocampal slice sGC activity. Nevertheless the potentiation, but not the preceding depression, was blocked by ODQ. l -nitroarginine and an NMDA antagonist were similarly effective, indicating mediation by the endogenous NMDA receptor,NO synthase,sGC pathway. At a concentration normally too low to affect synaptic transmission but sufficient to stimulate sGC (estimated to be 50 nm), exogenous NO reversed the effect of l -nitroarginine and caused a potentiation which was blocked by ODQ. At a concentration inducing the depression/potentiation sequence, NO partially inhibited hippocampal slice oxygen consumption. It is concluded that, at physiological levels, exogenous NO can directly elicit a potentiation of synaptic transmission through sGC, provided that the synapses are suitably primed. At higher concentrations, NO inhibits mitochondrial respiration, which can result in an enduring synaptic potentiation due to secondary activation of the endogenous NO,cGMP pathway. [source] A New and Productive Route to 1-HeteroarylcyclopropanolsEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 3 2003Vladimir N. Belov Abstract (E/Z)-2-(1-Allyloxycyclopropyl)-3-methoxyacrylonitrile (4 -All) was designed and prepared in five steps (58% overall yield) from ethyl cyclopropylidenacetate as a valuable precursor to various 1-heteroarylcyclopropanols. Its condensation with amidines, guanidine, hydrazine, and methyl thioglycolate and subsequent removal of the allyl protecting group yields 1-heteroarylcyclopropanols such as 1 -OH (36% over 2 steps), a very potent NO-independent stimulator of soluble guanylate cyclase. Direct cleavage of the allyl ether protecting group [by palladium-catalyzed substitution with lithium p -toluenesulfinate in AcOH or treatment with c -HexMgBr/Ti(OiPr)4] gives highly functionalized, sterically congested 1-heteroarylcyclopropanols 29, 30, and 34 with intact amino and ester groups. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003) [source] Homologous desensitization of guanylyl cyclase A, the receptor for atrial natriuretic peptide, is associated with a complex phosphorylation patternFEBS JOURNAL, Issue 11 2010Juliane Schröter Atrial natriuretic peptide (ANP), via its guanylyl cyclase A (GC-A) receptor and intracellular guanosine 3,,5,-cyclic monophosphate production, is critically involved in the regulation of blood pressure. In patients with chronic heart failure, the plasma levels of ANP are increased, but the cardiovascular actions are severely blunted, indicating a receptor or postreceptor defect. Studies on metabolically labelled GC-A-overexpressing cells have indicated that GC-A is extensively phosphorylated, and that ANP-induced homologous desensitization of GC-A correlates with receptor dephosphorylation, a mechanism which might contribute to a loss of function in vivo. In this study, tandem MS analysis of the GC-A receptor, expressed in the human embryonic kidney cell line HEK293, revealed unambiguously that the intracellular domain of the receptor is phosphorylated at multiple residues: Ser487, Ser497, Thr500, Ser502, Ser506, Ser510 and Thr513. MS quantification based on multiple reaction monitoring demonstrated that ANP-provoked desensitization was accompanied by a complex pattern of receptor phosphorylation and dephosphorylation. The population of completely phosphorylated GC-A was diminished. However, intriguingly, the phosphorylation of GC-A at Ser487 was selectively enhanced after exposure to ANP. The functional relevance of this observation was analysed by site-directed mutagenesis. The substitution of Ser487 by glutamate (which mimics phosphorylation) blunted the activation of the GC-A receptor by ANP, but prevented further desensitization. Our data corroborate previous studies suggesting that the responsiveness of GC-A to ANP is regulated by phosphorylation. However, in addition to the dephosphorylation of the previously postulated sites (Ser497, Thr500, Ser502, Ser506, Ser510), homologous desensitization seems to involve the phosphorylation of GC-A at Ser487, a newly identified site of phosphorylation. The identification and further characterization of the specific mechanisms involved in the downregulation of GC-A responsiveness to ANP may have important pathophysiological implications. Structured digital abstract ,,MINT-7713870, MINT-7713887: PMCA (uniprotkb:P20020) and GC-A (uniprotkb:P18910) colocalize (MI:0403) by fluorescence microscopy (MI:0416) [source] ATP allosteric activation of atrial natriuretic factor receptor guanylate cyclaseFEBS JOURNAL, Issue 11 2010Teresa Duda Atrial natriuretic factor receptor guanylate cyclase (ANF-RGC) is the receptor and the signal transducer of two natriuretic peptide hormones: atrial natriuretic factor and brain natriuretic peptide. It is a single transmembrane-spanning protein. It binds these hormones at its extracellular domain and activates its intracellular catalytic domain. This results in the accelerated production of cyclic GMP, a second messenger in controlling blood pressure, cardiac vasculature and fluid secretion. ATP is obligatory for the transduction of this hormonal signal. Two models of ATP action have been proposed. In Model 1, it is a direct allosteric transducer. It binds to the defined regulatory domain (ATP-regulated module) juxtaposed to the C-terminal side of the transmembrane domain of ANF-RGC, induces a cascade of temporal and spatial changes and activates the catalytic module residing at the C-terminus of the cyclase. In Model 2, before ATP can exhibit its allosteric effect, ANF-RGC must first be phosphorylated by an as yet unidentified protein kinase. This initial step is obligatory in atrial natriuretic factor signaling of ANF-RGC. Until now, none of these models has been directly validated because it has not been possible to segregate the allosteric and the phosphorylation effects of ATP in ANF-RGC activation. The present study accomplishes this aim through a novel probe, staurosporine. This unequivocally validates Model 1 and settles the over two-decade long debate on the role of ATP in ANF-RGC signaling. In addition, the present study demonstrates that the mechanisms of allosteric modification of ANF-RGC by staurosporine and adenylyl-imidodiphosphate, a nonhydrolyzable analog of ATP, are almost (or totally) identical. [source] The allene oxide cyclase family of Arabidopsis thaliana , localization and cyclizationFEBS JOURNAL, Issue 10 2008Florian Schaller Jasmonates are derived from oxygenated fatty acids (oxylipins) via the octadecanoid pathway and are characterized by a pentacyclic ring structure. They have regulatory functions as signaling molecules in plant development and adaptation to environmental stress. Recently, we solved the structure of allene oxide cyclase 2 (AOC2) of Arabidopsis thaliana, which is, together with the other three AOCs, a key enzyme in the biosynthesis of jasmonates, in that it releases the first cyclic and biologically active metabolite , 12-oxo-phytodienoic acid (OPDA). On the basis of models for the bound substrate, 12,13(S)-epoxy-9(Z),11,15(Z)-octadecatrienoic acid, and the product, OPDA, we proposed that a conserved Glu promotes the reaction by anchimeric assistance. According to this hypothesis, the transition state with a pentadienyl carbocation and an oxyanion is stabilized by a strongly bound water molecule and favorable ,,, interactions with aromatic residues in the cavity. Stereoselectivity results from steric restrictions to the necessary substrate isomerizations imposed by the protein environment. Here, site-directed mutagenesis was used to explore and verify the proposed reaction mechanism. In a comparative analysis of the AOC family from A. thaliana involving enzymatic characterization, in vitro import, and transient expression of AOC,enhanced green fluorescent protein fusion proteins for analysis of subcellular targeting, we demonstrate that all four AOC isoenzymes may contribute to jasmonate biosynthesis, as they are all located in chloroplasts and, in concert with the allene oxide synthase, they are all able to convert 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid into enantiomerically pure cis(+)-OPDA. [source] Irregular dimerization of guanylate cyclase-activating protein 1 mutants causes loss of target activationFEBS JOURNAL, Issue 18 2004Ji-Young Hwang Guanylate cyclase-activating proteins (GCAPs) are neuronal calcium sensors that activate membrane bound guanylate cyclases (EC 4.6.1.2.) of vertebrate photoreceptor cells when cytoplasmic Ca2+ decreases during illumination. GCAPs contain four EF-hand Ca2+ -binding motifs, but the first EF-hand is nonfunctional. It was concluded that for GCAP-2, the loss of Ca2+ -binding ability of EF-hand 1 resulted in a region that is crucial for targeting guanylate cyclase [Ermilov, A.N., Olshevskaya, E.V. & Dizhoor, A.M. (2001) J. Biol. Chem.276, 48143,48148]. In this study we tested the consequences of mutations in EF-hand 1 of GCAP-1 with respect to Ca2+ binding, Ca2+ -induced conformational changes and target activation. When the nonfunctional first EF-hand in GCAP-1 is replaced by a functional EF-hand the chimeric mutant CaM,GCAP-1 bound four Ca2+ and showed similar Ca2+ -dependent changes in tryptophan fluorescence as the wild-type. CaM,GCAP-1 neither activated nor interacted with guanylate cyclase. Size exclusion chromatography revealed that the mutant tended to form inactive dimers instead of active monomers like the wild-type. Critical amino acids in EF-hand 1 of GCAP-1 are cysteine at position 29 and proline at position 30, as changing these to glycine was sufficient to cause loss of target activation without a loss of Ca2+ -induced conformational changes. The latter mutation also promoted dimerization of the protein. Our results show that EF-hand 1 in wild-type GCAP-1 is critical for providing the correct conformation for target activation. [source] Molecular and functional characterization of novel CRFR1 isoforms from the skinFEBS JOURNAL, Issue 13 2004Alexander Pisarchik In our continued studies on corticotropin releasing factor receptor (CRFR1) signaling in the skin, we tested functional activity of CRFR1,, e, f, g and h isoforms after transfection to COS cells. Both membrane-bound and soluble variants are translated in vivo into final protein products that undergo further post-translational modifications. CRFR1, was the only isoform coupled directly to adenylate cyclase with the exception of an artificial isoform (CRFR1h2) with the insertion of 37 amino acids between the ligand binding domain and the first extracellular loop that was capable of producing detectable levels of cyclic AMP (cAMP). Soluble isoforms could modulate cell response with CRFR1e attenuating and CRFR1h amplifying CRFR1,-coupled cAMP production stimulated by urocortin. Testing with plasmids containing the luciferase reporter gene, and inducible cis -elements (CRE, CaRE, SRE, AP1 or NF-,B) demonstrated that only CRFR1, was involved directly in the transcriptional regulation, while CRFR1g inhibited CRE activity. Significantly higher reporter gene expression by CRF was observed than that mediated by 4,-phorbol 12-myristate 13-acetate and forskolin alone, being compatible with the concomitant treatment by phorbol 12-myristate 13-acetate and forskolin. This suggests that both protein kinase A and C can be involved in CRF-dependent signal transduction. [source] Fusion of farnesyldiphosphate synthase and epi -aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacumFEBS JOURNAL, Issue 14 2002Maria Brodelius A clone encoding farnesyl diphosphate synthase (FPPS) was obtained by PCR from a cDNA library made from young leaves of Artemisia annua. A cDNA clone encoding the tobacco epi -aristolochene synthase (eAS) was kindly supplied by J. Chappell (University of Kentucky, Lexington, KY, USA). Two fusions were constructed, i.e. FPPS/eAS and eAS/FPPS. The stop codon of the N-terminal enzyme was removed and replaced by a short peptide (Gly-Ser-Gly) to introduce a linker between the two ORFs. These two fusions and the two single cDNA clones were separately introduced into a bacterial expression vector (pET32). Escherichia coli was transformed with the expression vectors and enzymatically active soluble proteins were obtained after induction with isopropyl thio-,- d -thiogalactoside. The recombinant enzymes were purified using immobilized metal affinity chromatography on Co2+ columns. The fusion enzymes produced epi- aristolochene from isopentenyl diphosphate through a coupled reaction. The Km values of FPPS and eAS for isopentenyl diphosphate and farnesyl diphosphate, respectively, were essentially the same for the single and fused enzymes. The bifunctional enzymes showed a more efficient conversion of isopentenyl diphosphate to epi -aristolochene than the corresponding amount of single enzymes. [source] Cross-talk between olfactory second messenger pathwaysFEBS JOURNAL, Issue 14 2000Alexander Vogl The second messengers 3,-5,-cyclic-monophosphate (cAMP) and inositol 1,4,5-trisphosphate (InsP3) have been implicated in olfactory signal transduction in various species. The results of the present study provide evidence that the two olfactory second messenger pathways in rat olfactory neurons do not work independently but rather show a functional antagonism: whereas inhibition of phospholipase C (PLC) in isolated olfactory cilia by U-73122 led to an augmentation of odor-induced cAMP signaling, activation of the phosphoinositol pathway resulted in attenuation of odor-induced cAMP formation. Furthermore, this study indicates that elevated cAMP levels cause suppression of odor-induced InsP3 signaling, whereas inhibition of adenylate cyclase (AC) by cisN -(2-phenylcyclopentyl)azacylotridec-1-en-2-amine (MDL-12,330 A) results in potentiation of odor-induced InsP3 formation. Concerning the molecular mechanism involved in cross-interaction, the experimental data indicate that the observed antagonism of elevated cAMP is based on inhibition of PLC activation rather than on stimulation of InsP3 degradation. As blockage of the endogenous protein kinase A (PKA) prevented the inhibitory effect of cAMP, the suppression of odor-induced InsP3 signaling by cAMP may be mediated by a PKA-controlled reaction. [source] The P2Y1 receptor mediates ADP-induced p38 kinase-activating factor generation in human plateletsFEBS JOURNAL, Issue 8 2000Carol Dangelmaier U46619, a thromboxane A2 mimetic, but not ADP, caused activation of p38 mitogen activated protein (MAP) kinase in aspirin-treated platelets. In nonaspirinated human platelets ADP activated p38 MAP kinase in both a time-and concentration-dependent manner, suggesting that ADP-induced p38 MAP kinase activation requires generation of thromboxane A2. However, neither a thromboxane A2/prostaglandin H2 receptor antagonist SQ29548 and a thromboxane synthase inhibitor, furegrelate, either alone or together, nor indomethacin blocked ADP-induced p38 kinase activation in nonaspirinated platelets. Other cycloxygenase products, PGE2, PGD2, and PGF2,, failed to activate p38 kinase in aspirin-treated platelets. Hence, ADP must be generating an agonist, other than thromboxane A2, via an aspirin-sensitive pathway, which is capable of activating p38 kinase. AR-C66096, a P2TAC (platelet ADP receptor coupled to inhibition of adenylate cyclase) antagonist, did not inhibit ADP-induced p38 MAP kinase activation. The P2X receptor selective agonist, ,,,-methylene ATP, failed to activate p38 MAP kinase. On the other hand, the P2Y1 receptor selective antagonist, adenosine-2,-phosphate-5,-phosphate inhibited ADP-induced p38 kinase activation in a concentration-dependent manner, indicating that the P2Y1 receptor alone mediates ADP-induced generation of the p38 kinase-activating factor. These results demonstrate that ADP causes the generation of a factor in human platelets, which can activate p38 kinase, and that this response is mediated by the P2Y1 receptor. Neither the P2TAC receptor nor the P2X1 receptor has any significant role in this response. [source] | ||||||||||||||||||||||||||||||||||