Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Adenosine and adenosine analogues are potent inhibitors of the respiratory burst in neutrophils. Most investigators, however, have found little or no effect of these compounds on neutrophil degranulation from cytochalasin B-treated neutrophils in suspension. We have instead investigated the effect of adenosine and 2-chloroadenosine on degranulation in adherent neutrophils in the absence of cytochalasin B. Both adenosine and 2-chloroadenosine were effective inhibitors of lactoferrin secretion induced by the chemotactic peptide N-formyl-methionine-leucyl-phenylalanine (fMLP) [50% inhibitory concentration (IC50) of less than 10(-6) M]. Secretion induced by tumor necrosis factor (TNF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) was inhibited only at high concentrations (IC50 of approximately 10(-4) M). In the presence of cytochalasin B no inhibitory effect of 2-chloroadenosine was seen. The effect of cAMP-raising agents on secretion from adherent neutrophils was also investigated. Dibutyryl cAMP at 0.2 mM reduced secretion in response to fMLP by 50% but did not inhibit TNF- and GM-CSF-induced degranulation. At a concentration of 2.0 mM dibutyryl cAMP also inhibited exocytosis in response to the two cytokines. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) at 300 microM reduced fMLP-induced degranulation, whereas a concentration of 1 mM was required to inhibit TNF- and GM-CSF-mediated secretion. The adenylate cyclase activator forskolin (50 microM) alone did not inhibit secretion in response to TNF or fMLP. However, in combination with IBMX (300 microM), forskolin (50 microM) reduced both TNF- and fMLP-induced secretion to less than 10%. PMA-induced exocytosis was unaffected by all these agents. In conclusion, adenosine appears to be an effective inhibitor of neutrophil granule protein secretion induced by fMLP but only a weak inhibitor of exocytosis in response to TNF or GM-CSF. Secretion in response to fMLP was also found to be more susceptible to a rise in cAMP than degranulation induced by TNF and GM-CSF.
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PMID:Effect of adenosine analogues and cAMP-raising agents on TNF-, GM-CSF-, and chemotactic peptide-induced degranulation in single adherent neutrophils. 137 3

Exposure to the phorbol ester, phorbol 12-myristate, 13-acetate (PMA, 100nM) for 10 minutes enhanced cyclic AMP accumulation in human neutrophils under basal conditions and in response to the beta-adrenergic receptor agonist isoproterenol (ISO), 1 microM) and the adenylate cyclase activator forskolin (FSK, 10mM). Potentiation of responses to ISO by PMA was dose-dependent between 0.1 and 100nM PMA. The diacylglycerol analogue, 1-oleoyl-2-acetylglycerol (OAG) (50 microM) also elevated beta-receptor responses, but 4 beta-phorbol (100nM), lacking the capacity to activate PMA, was ineffective. Short-term exposure (12 seconds) to the peptide n-formylmethionine leucyl-phenylalanine (FMLP, 1 microM) also elevated neutrophil cyclic AMP accumulation. All potentiating effects of PMA on cyclic AMP production were inhibited by the protein kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7). Elevation of cyclic AMP by FMLP was insensitive to H7. PMA had no apparent effect on beta-receptor agonist-affinity, distribution between cell-surface and internalised compartments, or the capacity of ISO to induce beta-receptor internalisation. Responses to FSK or ISO in terms of fold-stimulation of basal cyclic AMP accumulation in the presence of PMA were not elevated by PMA. These findings indicate that PMA exerts a potentiating effect on neutrophil adenylate cyclase responses through protein kinase C activation. FMLP elevation of neutrophil cyclic AMP in the absence of other stimuli, appears however, to be insensitive to protein kinase inhibition.
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PMID:Neutrophil beta-adrenergic receptor responses are potentiated by acute exposure to phorbol ester without changes in receptor distribution or coupling. 165 46

The alpha subunits of Gi (Gi alpha) and Gs (guanine-nucleotide-binding proteins involved in adenylate cyclase inhibition and stimulation, respectively) was ADP-ribosylated by cholera toxin in differentiated HL-60 cell membranes upon stimulation of chemotactic receptors by fMLF (fM, N-formylmethionine). The ADP-ribosylation site of Gi alpha modified by cholera toxin appeared to be different from that modified by pertussis toxin [Iiri, T., Tohkin, M., Morishima, N., Ohoka, Y., Ui, M. & Katada, T. (1989) J. Biol. Chem. 264, 21,394-21,400]. This allowed us to investigate how the two types of ADP-ribosylation influence the function of the signal-coupling protein. The major findings observed in HL-60 cell membranes, where the same Gi alpha molecule was ADP-ribosylated by treatment of the membranes with either toxin, are summarized as follows. (a) More fMLF bound with a high affinity to cholera-toxin-treated membranes than to the control membranes. The high-affinity binding was, however, not observed in pertussis-toxin-treated membranes. (b) Although fMLF stimulated guanine nucleotide binding and GTPase activity in control membranes, stimulation was almost completely abolished in pertussis-toxin-treated membranes. In contrast, fMLF-dependent stimulation of GTPase activity, but not that of guanine nucleotide binding was attenuated in cholera-toxin-treated membranes. (c) Gi alpha, once modified by cholera toxin, still served as a substrate of pertussis-toxin-catalyzed ADP-ribosylation; however, the ADP-ribosylation rate of modified Gi was much lower than that of intact Gi. These results suggested that Gi ADP-ribosylated by cholera toxin was effectively capable of coupling with fMLF receptors, resulting in formation of high-affinity fMLF receptors, and that hydrolysis of GTP bound to the alpha subunit was selectively impaired by its ADP-ribosylation by cholera toxin. Thus, unlike the ADP-ribosylation of Gi by pertussis toxin, cholera-toxin-induced modification would be of great advantage to the interaction of Gi with receptors and effectors that are regulated by the signal-coupling protein. This type of modification might also be a candidate for unidentified G proteins which were less sensitive to pertussis toxin and appeared to be involved in some signal-transduction systems.
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PMID:Functional modification by cholera-toxin-catalyzed ADP-ribosylation of a guanine-nucleotide-binding regulatory protein serving as the substrate of pertussis toxin. 166 35

Considerable evidence suggests that signal transduction pathways are targets of lithium (Li) action. A number of investigators have reported that Li attenuates both adenylate cyclase (AC) activity and phosphoinositide (PI) turnover in rodents and in humans, thus "dampening" these systems. We have studied selected components of these second-messenger systems in a series of clinical and preclinical investigations. To overcome confounding effects of alterations in mood state, we examined AC activity and G-protein ribosylation in peripheral blood cells from 10 healthy volunteers, prior to and following 14 days of Li administration. Basal and postreceptor [cesium fluoride (CsF) or Gpp(NH)p] stimulated AC activity were unaffected in lymphocytes. In contrast, both basal and stimulated AC activity in platelets were significantly augmented, compatible with an attenuation of Gi function. Ribosylation of platelet Gs by cholera toxin was unchanged, whereas that of Gi by pertussis toxin (PT) was increased. Given that undissociated G protein is the preferred substrate for PT, our results suggest that Li interferes with subunit dissociation and the subsequent activation of Gi. To determine if Li has similar effects on Gi in the central nervous system, we measured extracellular (EC) cyclic adenosine monophosphate (cAMP) in rat brain by in vivo microdialysis, revealing a dose-dependent increase in cAMP by norepinephrine (NE) antagonized by propranolol. Chronic (4-week) Li doubled basal EC cAMP, while decreasing the fractional response to 100 microM NE. Thus, using in vivo microdialysis, we observed the reported reduction in NE-stimulated AC activity, but only as a function of elevated basal cAMP. Increased basal AC activity has been observed following chronic Li in both humans and rat tissues but generally has not been considered relevant. The PI generating system is another proposed major target for Li that we have studied using an in vitro cell culture model of peripheral blood cells. Chronic (6-day) exposure of neutrophil-like HL60 cells to 1 mM LiCl did not affect agonist fMet-Leu-Phe (fMLP) induced PI turnover. In contrast, Li attenuated both agonist and phorbol ester stimulated Na+/H+ exchange, suggesting reduced protein kinase C (PKC) function. Western blot analysis revealed altered levels of PKC in both membrane and cytosolic fractions. The functional consequences of these complex effects on the two major signal transduction pathways and their interactions in the intact living organism remain to be elucidated.
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PMID:Signal transduction modulation by lithium: cell culture, cerebral microdialysis and human studies. 177 89

Upon exposure to the bacterial chemotactic peptide fMet-Leu-Phe, human neutrophils release lysozyme and generate superoxide anions (O2.-). The synthetic lipoamino acid N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam3Cys), which is derived from the N-terminus of bacterial lipoprotein, when attached to Ser-(Lys)4 [giving Pam3Cys-Ser-(Lys)4], activated O2.- formation and lysozyme release in human neutrophils with an effectiveness amounting to about 15% of that of fMet-Leu-Phe. Palmitic acid, muramyl dipeptide, lipopolysaccharide and the lipopeptides Pam3Cys-Ala-Gly, Pam3Cys-Ser-Gly, Pam3Cys-Ser, Pam3Cys-OMe and Pam3Cys-OH did not activate O2.- formation. Pertussis toxin, which ADP-ribosylates guanine-nucleotide-binding proteins (G-proteins) and functionally uncouples formyl peptide receptors from G-proteins, prevented activation of O2.- formation by fMet-Leu-Phe and inhibited Pam3Cys-Ser-(Lys)4-induced O2.- formation by 85%. Lipopeptide-induced exocytosis was pertussis-toxin-insensitive. O2.- formation induced by Pam3Cys-Ser-(Lys)4 and fMet-Leu-Phe was enhanced by cytochalasin B, by a phorbol ester and by a diacylglycerol kinase inhibitor. Addition of activators of adenylate cyclase and removal of extracellular Ca2+ inhibited O2.- formation by fMet-Leu-Phe and Pam3Cys-Ser-(Lys)4 to different extents. Pam3Cys-Ser-(Lys)4 synergistically enhanced fMet-Leu-Phe-induced O2.- formation and primed neutrophils to respond to the chemotactic peptide at non-stimulatory concentrations. Our data suggest the following. (1) Pam3Cys-Ser-(Lys)4 activates neutrophils through G-proteins, involving pertussis-toxin-sensitive and -insensitive processes. (2) The signal transduction pathways activated by fMet-Leu-Phe and Pam3Cys-Ser-(Lys)4 are similar but not identical. (3) In inflammatory processes, bacterial lipoproteins and chemotactic peptides may interact synergistically to activate O2.- formation, leading to enhanced bactericidal activity.
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PMID:Activation of superoxide formation and lysozyme release in human neutrophils by the synthetic lipopeptide Pam3Cys-Ser-(Lys)4. Involvement of guanine-nucleotide-binding proteins and synergism with chemotactic peptides. 216 Feb 37

Recombinant analogues of human parathyroid hormone [hPTH-(1-84)] were expressed in Escherichia coli harboring plasmids containing synthetic genes under the control of the lac promoter. The level of expression of the gene encoding the truncated analogue, hPTH-(3-84), was greater than that of the gene encoding full-length hPTH-(1-84) but less than that of the gene encoding proparathyroid hormone (hProPTH). This may be due in part to the relative efficiency of translation of the mRNA as suggested by secondary structure analysis and in part because of enhanced stability of the extended peptide. Formylmethionyl derivatives of hProPTH and of hPTH-(3-84) and underivatized hPTH-(3-84) were purified by HPLC, and their identity was confirmed by NH2-terminal sequencing and amino acid analysis. The bioactivity of these recombinant peptides was then tested in skeletal and renal adenylate cyclase assays in vitro and in assays examining effects on plasma and urine calcium and phosphate levels and on urine cyclic AMP levels in vivo. The NH2-terminally extended analogue fMet-hProPTH displayed 10% of the in vitro activity of hPTH-(1-84) and was a partial agonist in vivo. The peptides hPTH-(3-84) and fMet-hPTH-(3-84) were inert in vitro and were very weak in vitro antagonists when compared to the NH2-terminal analogue bovine [Nle8,18Tyr34]PTH-(3-34)-NH2. In vivo, hPTH-(3-84) and the bPTH-(3-34) analogue, when assayed at a 10:1 molar ratio relative to bPTH-(1-84), were each inert, and neither demonstrated antagonist activity at these concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Synthesis and characterization of extended and deleted recombinant analogues of parathyroid hormone-(1-84): correlation of peptide structure with function. 217 61

Human parathyroid hormone (hPTH) is a peptide hormone consisting of 84 amino acids. Using the expression plasmid pKK223-3 with the strong tacpromoter, we have produced a variant of hPTH in E. coli. From the expression plasmid construct the expected product was hPTH with an N-terminal extension of Met-Gly. The peptide was extracted from E. coli cells and purified by high performance liquid chromatography. In two different gel electrophoresis systems including identification by immunoblotting the product behaved exactly as an hPTH standard. N-terminal amino acid sequence analysis of the purified product showed traces of Gly-hPTH. At least 90% of the expressed product was N-terminally blocked, suggesting the presence of N-formyl-methionine. This variant of hPTH did not stimulate adenylate cyclase activity in rat osteosarcoma cell membranes.
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PMID:Expression of human parathyroid hormone in Escherichia coli. 240 51

We have investigated the sequence of events leading from the activation of adenylate cyclase and increases in intracellular cyclic AMP to the modulation of enzyme release and superoxide production in human neutrophils. In the isolated plasma membrane, adenylate cyclase is activated by both prostaglandin E1 and isoproterenol. In the whole cell only a small increase in cyclic AMP is observed, though in the presence of the phosphodiesterase inhibitor, methylisobutylxanthine a substantial amplification in intracellular cyclic AMP is observed with both isoproterenol and prostaglandin E1. These conditions are relevant to the regulation of cell function, since fMet-Leu-Phe-stimulated superoxide production is inhibited by either prostaglandin E1 or isoproterenol in the absence of methylisobutylxanthine, while enzyme release is inhibited only via the prostaglandin E1 receptor and then only in the presence of methylisobutylxanthine. For enzyme release and superoxide production, the order of potency for three prostaglandins tested was prostaglandin E1 greater than prostaglandin D2 much greater than prostaglandin F2 alpha. Our results suggest that (a) superoxide production is more sensitive to regulation by cyclic AMP than enzyme release, (b) the type of receptor occupied as well as the threshold level of cyclic AMP attained are important to the regulation of enzyme release, and (c) although elevation in cyclic AMP is inhibitory to neutrophil function, phosphodiesterase inhibition is required in addition to adenylate cyclase activation to effect maximal inhibition.
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PMID:Receptor-specific threshold effects of cyclic AMP are involved in the regulation of enzyme release and superoxide production from human neutrophils. 241 Dec 98

Human neutrophils and HL-60 leukaemic cells possess an NADPH oxidase which catalyses superoxide (O2-) formation and is activated by the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe). In dibutyryl cyclic AMP-differentiated HL-60 cells, ATP and UTP in the presence of cytochalasin B activated O2- formation with EC50 values of 5 microM and efficacies amounting to 30% of that of fMet-Leu-Phe. The potency order of purine nucleotides in activating O2- generation was ATP = adenosine 5'-O-(3-thiotriphosphate) greater than ITP greater than dATP = ADP. Pyrimidine nucleotides activated NADPH oxidase in the potency order UTP greater than dUTP greater than CTP = TTP = UDP. Pertussis toxin completely prevented activation of NADPH oxidase by fMet-Leu-Phe and UTP, whereas the effect of ATP was only partially inhibited. ATP and UTP enhanced O2- generation induced by fMet-Leu-Phe by up to 8-fold, and primed the cells to respond to non-stimulatory concentrations of fMet-Leu-Phe. Activation of NADPH oxidase by UTP but not by ATP was inhibited by various activators of adenylate cyclase. In dimethyl sulphoxide-differentiated HL-60 cells and in human neutrophils, ATP and UTP per se did not activate NADPH oxidase, but they potentiated the effect of fMet-Leu-Phe. Our results suggest that purine and pyrimidine nucleotides act via purino- and novel pyrimidinoceptors respectively, which are coupled to guanine nucleotide-binding proteins leading to the activation of NADPH oxidase. As ATP and UTP are released from cells under physiological and pathological conditions, these nucleotides may play roles as intercellular signal molecules in the activation of O2- formation.
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PMID:Activation of NADPH oxidase by purine and pyrimidine nucleotides involves G proteins and is potentiated by chemotactic peptides. 254 70

In order to analyze the complex activities of histamine H2 receptor activation on neutrophils, human HL-60 promyelocytic leukemia cells were differentiated into neutrophils by incubation with dimethyl sufoxide, loaded with the Ca2+-sensitive indicator dyes, indo-1 or fura-2, and the levels of intracellular Ca2+ ([Ca2+]i) measured in a fluorescent-activated cell sorter and fluorimeter, respectively. Histamine increased [Ca2+]i in a dose-dependent manner with a half-maximal concentration (EC50) of approximately 10(-6) to 10(-5) M, which exhibited H2 receptor specificity. Prostaglandin E2 and isoproterenol also induced [Ca2+]i mobilization in HL-60 cells, whereas the cell permeable form of cAMP and forskolin failed to increase [Ca2+]i. Since H2-receptor mediated [Ca2+]i mobilization was not inhibited by reducing the concentration of extracellular Ca2+ nor by the addition of Ca2+ channel antagonists, LaCl3 and nifedipine, [Ca2+]i mobilization is due to the release of Ca2+ from intracellular stores. Furthermore, both 10(-4) M histamine and 10(-6) M fMet-Leu-Phe increased the levels of 1,4,5-inositol trisphosphate. However, histamine-induced mobilization of [Ca2+]i was inhibited by cholera toxin but not by pertussis toxin, whereas the action of fMet-Leu-Phe was inhibited by pertussis toxin but not by cholera toxin. These data suggest that H2 receptors on HL-60 cells are coupled to two different cholera toxin-sensitive G-proteins and activate adenylate cyclase and phospholipase C simultaneously.
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PMID:Multiple signaling pathways of histamine H2 receptors. Identification of an H2 receptor-dependent Ca2+ mobilization pathway in human HL-60 promyelocytic leukemia cells. 255 5


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