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)

Prenalterol, an allegedly beta 1-selective adrenergic agonist with high intrinsic sympathomimetic activity (ISA), was shown to be weakly lipolytic in rat adipocytes. However, in pertussis-toxin-treated adipocytes, the ISA of prenalterol was markedly increased (from 10-20% to approx. 100% of that of isoprenaline). The cellular sensitivity was also increased (EC50 approx. 60 nM and approx. 3 microM in pertussis-toxin-treated and control cells respectively). A similar effect was seen for other partial agonists such as the beta 2-selective agonist terbutaline and for beta-adrenergic antagonists with some intrinsic activity (metoprolol, pindolol). There was no clear change in sensitivity to isoprenaline's ability to stimulate adenylate cyclase in adipocyte membranes from pertussis-toxin-treated animals but the cyclase activity was increased approx. 4-fold in the presence of 1 microM-GTP. Prenalterol stimulated lipolysis by only small increases in intracellular cyclic AMP (cAMP) levels (less than 10% of that seen with isoprenaline). Basal lipolysis was increased in cells from pertussis-toxin-treated rats and the cellular sensitivity to the non-degradable cAMP analogue, N6-monobutyryl-cAMP, was increased. In control cells, a submaximal concentration of prenalterol (0.1 microM) increased the sensitivity to the cAMP analogues, N6-monobutyryl-cAMP and 8-bromo-cAMP. A low concentration (1 mM) of 8-bromo-cAMP also increased the effect of prenalterol. Similar effects were seen when the phosphodiesterase was inhibited. Thus (1) lipolysis is extremely sensitive to small increases in intracellular cAMP; (2) the degree of activation of adenylate cyclase and thus cAMP formation is the rate-limiting step for the biological response of partial agonists; (3) the inhibitory GTP-binding protein, Gi, is an important modulator ('tissue factor') of the beta-adrenergic agonistic property; (4) low levels of cAMP exert a priming effect on protein kinase A.
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PMID:The inhibitory GTP-binding protein (Gi) regulates the agonistic property of beta-adrenergic ligands in isolated rat adipocytes. Evidence for a priming effect of cyclic AMP. 128 Jan 15

Growth and differentiation of cells derived from the embryonic palate are critically dependent on the intracellular cAMP-mediated signal transduction pathway. Human embryonic palate mesenchymal (HEPM) cells have been widely used to examine the effect of teratogens on palatal tissue growth and differentiation, as well as a prescreen for environmental teratogens. This study examined responsiveness of HEPM cells to agents known to stimulate adenylate cyclase, characterized cAMP-dependent protein kinases (cAMP-dPK) (EC 2.7.1.37) and investigated to what extent HEPM cells reveal adaptational responses to cAMP at the level of cAMP-dependent protein kinase. HEPM cells exhibited a total cell cycle transit time of approximately 22 h and responded maximally, when confluent, to prostacyclin (PGI2), prostaglandin E2 (PGE2), and isoproterenol with time- and dose-dependent increases in intracellular levels of cAMP. The order of sensitivity to hormonal activation of adenylate cyclase was PGE2 > isoproterenol > PGI2. Basal cAMP-dependent protein kinases activity was 0.184 fmol phosphate transferred from ATP to histone per microgram protein per minute under conditions where endogenous phosphatases did not significantly affect protein phosphorylation. Regulatory subunits of cAMP-dPK in HEPM cells were characterized by the binding of [3H]cAMP to cytosolic fractions. Specific binding was saturable at approximately 50 nM indicating the presence of binding sites that are finite in number. Calculation of half-maximal binding yielded an estimated Kd of 25 nM indicating the presence of high affinity binding sites. Cyclic AMP-dPK regulatory subunits were also photoaffinity labeled with 8-N3-[32P]-cAMP, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and radiolabeled bands visualized by autoradiography. Photoactivated incorporation of 8-N3-[32P]cAMP was detected into two proteins of molecular weight (M(r)) 45,000 and M(r) 51,000 representing, respectively, the RI alpha and RII beta subunits of cAMP-dPK. Binding of [32P]8-azido cAMP to proteins of M(r) 45,000 (RI alpha) and M(r) 51,000 (RII beta) was increased in response to elevation of intracellular cAMP via inhibition of its breakdown with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, or by direct activation of adenylate cyclase with forskolin. HEPM cells thus revealed adaptational responses to cAMP at the level of cAMP-dependent protein kinase. Characterization of the cAMP signal transduction pathway in HEPM cells, derived from embryonic palatal tissue which is critically dependent on this pathway for normal development, may provide information fundamental to a clear understanding of cellular events involved in palatal ontogeny. These results highlight several important differences between HEPM cells and murine embryonic palate mesenchymal cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Cyclic AMP-dependent protein kinase in human embryonic palate mesenchymal cells. 128 15

Endogenous calmodulin (CaM) in the EGTA-washed cerebral-cortical synaptosomal membrane (SM) preparation was estimated below 3 micrograms/ml protein by the semiquantitative immunoblot analysis (Natsukari, N., Ohta, H. and Fujita, M. (1989) J. Immunol. Methods 125, 159-166). Membrane-bound CaM was immunoelectron-microscopically demonstrated in EGTA-washed, non-treated (control), and Ca(2+)-treated cerebral-cortical synaptosomal membranes (SM) as well as for the SM enriched with added CaM. The density of CaM increased in the above order. CaM-dependent adenylate cyclase and CaM-dependent protein kinase II (CaM-kinase II) activities were restored, whereas the phosphodiesterase (PDE) activity was not affected by exogenous CaM over all the Ca2+ concentrations tested. Adenylate cyclase at pCa 6.2 was synergistically activated either by GTP and CaM or by CaM and beta-adrenergic agonist, (+/-)-isoproterenol, reflecting the intactness of signal transduction pathway in the SM. Also demonstrated were the presence of protein kinase A, CaM-kinase II, and their endogenous substrates in the SM. Based on 32P-autoradiography and 125I-CaM overlay data certain CaM-binding proteins such as CaM-kinase II and synapsin I were identified on SDS-PAGE. Ca(2+)-dependent and -independent CaMBPs were distinguished by 125I-CaM gel overlay with and without Ca2+. The former had bigger molecular size (greater than or equal to 49 kDa) than the latter (less than or equal to 34 kDa). Yield of Ca(2+)-dependent CaMBPs was not affected by Ca2+ concentration during preparation of the SM while that of Ca(2+)-independent CaMBPs was reduced by exposure to 100 microM Ca2+. In contrast with the CaMBPs of brain SM, those of enterocyte and eyrthrocyte plasma membranes especially, microvillous membrane of the enterocyte, showed quite distinct CaMBP profiles. The present findings suggested that the EGTA-washed SM preparation made a useful system for studying the role of CaM in the brain SM.
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PMID:Characterization of EGTA-washed synaptosomal membrane with emphasis on its calmodulin-binding proteins. Demonstration of possible reconstitution with added calcium/calmodulin. 131 53

Staurosporine, a protein kinase (PK) inhibitor, phorbol-12-myristate-13-acetate (PMA), a PKC activator and A23187 calcium ionophore were added to human melanocyte cultures with or without dibutyryl cyclic AMP (dbcAMP). After 2 days' incubation, changes in various melanogenic factors were examined such as tyrosinase activity and the amount of tyrosinase-related protein (TRP) as well as the morphology of the melanocytes. dbcAMP stimulated all the melanogenic factors. Staurosporine increased tyrosinase activity and amount of TRP and caused morphological changes with the formation of numerous dendrites, regardless of the presence of dbcAMP. In contrast, PMA did not significantly affect tyrosinase activity, TRP content or dendrite formation, with or without dbcAMP. The effects of staurosporine on tyrosinase activity and TRP content were completely inhibited by PMA, but PMA did not significantly affect the staurosporine-induced morphological changes. A23187 inhibited both tyrosinase activity and TRP content, regardless of the presence of dbcAMP, but did not affect the morphology of melanocytes. These findings suggest that tyrosinase activity and TRP content are regulated by adenylate cyclase and Ca2+ and partly by PKC, while the morphological features of melanocytes are affected by intracellular cAMP accumulation and by the inhibition of PKC.
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PMID:Effects of staurosporine, PMA and A23187 on human melanocyte cultures with dibutyryl cyclic AMP. 131 Nov 91

In this study, the role of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) in cAMP-dependent relaxation was assessed in the isolated-perfused rat lung using a PKA inhibitor, Rp-cAMPS, 8-bromo-cAMP (8-BrcAMP), and the diterpene activator of adenylate cyclase (AC), forskolin (FSK). A role for K+ channels was also assessed with the nonselective K+ channel blocker, tetraethylammonium (TEA, 10 mM), and an ATP-sensitive K+ channel inhibitor, glibenclamide (GLI, 100 microM). Both 8-BrcAMP (0.1-1.0 mM) and RSK (0.1-10 microM) dose-dependently attenuated the peak pressor response to alveolar hypoxia (HPR). Rp-cAMPS potentiated the HPR and attenuated 8-BrcAMP-mediated vasodilation but had no effect on FSK-mediated vasodilation. FSK-mediated vasodilation was not mimicked by 1,9-dideoxy-FSK, which is biologically inactive on AC but alters K+ channels identically to FSK, nor was it attenuated by the platelet-activating factor antagonist SRI 63-441 or the cyclooxygenase inhibitor indomethacin. TEA, but not GLI, attenuated FSK-mediated vasodilation. Similarly, TEA attenuated 8-BrcAMP-mediated vasodilation. These results support roles for PKA and indirect gating of a non-ATP-sensitive K+ channel in mediating cAMP-dependent pulmonary vasodilation.
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PMID:Role of cAMP-dependent protein kinase in cAMP-mediated vasodilation. 131 30

The factors which regulate the expression of the granin family of secretory proteins have yet to be completely described. The present study investigated the effects of forskolin (FSK), an activator of adenylate cyclase, on the regulation of chromogranin B/secretogranin I (CgB) and secretogranin II (SgII) mRNA levels in rat PC12 cells. PC12 cells were treated with 10 microM FSK for time points up to 48 h and were harvested for cAMP determination, RNA isolation and Northern blot analysis, or fixed in 4% paraformaldehyde for immunocytochemistry. Cellular cAMP levels peaked after two h of FSK treatment and remained elevated for 48 h. Chromogranin B mRNA increased with FSK treatment, reaching a maximum of 7-fold above control after 24 h, while the level of SgII mRNA decreased to a level of 65 +/- 10% of control after 48 h. The effects of FSK on CgB mRNA appear to be mediated by cAMP, as 8-bromo-cAMP (500 microM) resulted in a 2.8-fold increase in CgB mRNA, and H-89 (30 microM), a selective inhibitor of cAMP-dependent protein kinase, reduced the FSK-mediated response. The level of CgB was also increased in FSK-treated cells, as evidenced by immunofluorescent analysis which showed a more intense staining in PC12 cells treated with FSK for 48 h than in untreated cells. The intensity of SgII staining was diminished by FSK treatment, most likely a result of a decreased rate of synthesis as well as an increase in the release of SgII. This study demonstrated that the mRNA and protein levels of CgB and SgII are differentially regulated by cAMP in PC12 cells.
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PMID:Differential regulation of chromogranin B/secretogranin I and secretogranin II by forskolin in PC12 cells. 131 1

Parathyroid hormone action on renal proximal tubule function involves phospholipase C/protein kinase C as well as adenylate cyclase/protein kinase A mediated regulatory pathways. Tissue culture experiments suggest that low concentrations of PTH affect preferentially the phospholipase C/protein kinase C pathway. In vivo, both regulatory cascades are probably involved in the regulation of proximal tubule function. It is not clear at present whether the two intracellular pathways are linked to one or two PTH receptors. A polarized distribution of PTH receptor(s) involving different second messengers appears possible in proximal tubule epithelial cells. High-affinity (Kd 10(-11)-10(-12) M) PTH receptors in the range of circulating PTH concentrations in vivo remain to be identified. Structural and functional characterization of PTH receptors as well as of the PTH-sensitive intracellular mediators and transport systems form the basis for a better understanding of PTH-dependent regulation of proximal tubule function.
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PMID:Parathyroid hormone receptors in control of proximal tubule function. 131 47

Among the important pleiotropic responses to gamma interferon (IFN-gamma) during the activation of macrophages (M phi) is the increased expression of major histocompatibility complex class II genes. In the present study, infection with Leishmania donovani was shown to inhibit in parallel the induction by IFN-gamma of H-2 A beta gene transcription, class II mRNA accumulation, and H-2 Ad protein expression in cells of the murine macrophage cell line P388D1. Treatment of P388D1 cells with either the adenylate cyclase activator cholera toxin or the protein kinase A activator N6-2'-O-dibutyryl cyclic AMP (dibutyryl cAMP) similarly inhibited the induction by IFN-gamma of class II protein expression, and in parallel with Leishmania infection, cholera toxin inhibited the induction of mRNA for the H-2 A alpha and H-2 A beta proteins. Concentrations of intracellular cAMP were significantly increased in cholera toxin-treated cells but not in leishmania-infected cells. These findings indicate that at least one mechanism by which Leishmania infection attenuates the activation of M phi by IFN-gamma involves selective, transcriptional inhibition of major histocompatibility complex class II genes via a cAMP-independent mechanism.
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PMID:Inhibition of expression of major histocompatibility complex class II molecules in macrophages infected with Leishmania donovani occurs at the level of gene transcription via a cyclic AMP-independent mechanism. 131 26

Cross-regulation from the stimulatory to the inhibitory adenylylcyclase pathways has been described (Hadcock, J. R., Ros, M., Watkins, D. C., and Malbon, C. C. (1990) J. Biol. Chem. 265, 14784-14790). More recently, persistent activation (48 h) of the inhibitory adenylylcyclase pathway has been shown to cross-regulate the stimulatory pathway (i) enhancing the maximal response of beta-adrenergic agonits, (ii) increasing the expression of beta-adrenergic receptor, and (iii) reducing the ED50 for the isoproterenol-stimulated response by 50-fold (Hadcock, J. R., Port, J. D., and Malbon, C. C. (1991) J. Biol. Chem. 266, 11915-11922). Here, we report that short term activation (60 min) of the inhibitory adenylylcyclase pathway of hamster smooth muscle DDT1MF-2 cells with the A1-adenosine receptor agonist N6-phenylisopropyladenosine (PIA) likewise enhances the stimulatory adenylylcyclase response to the beta-adrenergic agonist isoproterenol. The PIA effect was exerted at the level of the receptor, i.e., the beta-adrenergic receptor-mediated response was enhanced, whereas the guanosine 5'-O-(thiotriphosphate)- and forskolin-stimulated adenylylcyclase activities were largely unaffected. In contrast to longer term persistent activation of the inhibitory pathway, receptor number and affinity for 125I-labeled cyanopindolol were unaffected. Metabolic labeling of cells with [32P]orthophosphate and immuneprecipitation of beta-adrenergic receptors detected phosphorylation of the receptor in unstimulated cells and marked phosphorylation in cells challenged with epinephrine. When cells were challenged short term with PIA, the basal state of beta-adrenergic receptor phosphorylation was reduced by 75%. Treating cells with PIA in combination with the cAMP analog 8-(4-chlorophenylthio)adenosine cyclic AMP attenuated the enhanced receptor-mediated adenylylcyclase response observed in cells treated with PIA alone. These data suggest that short term cross-regulation from the inhibitory to stimulatory adenylylcyclase pathways results in the following: (i) decreased intracellular cAMP levels and protein kinase A activity, (ii) reduced phosphorylation of the beta 2-adrenergic receptor in the "basal" (i.e. unstimulated) state, and (iii) enhanced receptor-mediated activation of Gs.
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PMID:Cross-regulation between G-protein-mediated pathways. Acute activation of the inhibitory pathway of adenylylcyclase reduces beta 2-adrenergic receptor phosphorylation and increases beta-adrenergic responsiveness. 131 28

We have investigated the effects of isoproterenol (ISO) and forskolin on carbachol(CCh)- and fluoroaluminate (AlF4-)-induced phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, myo-inositol 1,4,5-trisphosphate (IP3) production, 1,2-diacylglycerol, measured as phosphatidic acid (PA) formation, and contraction in the bovine iris sphincter smooth muscle. The data from these studies can be summarized as follows. (1) CCh (20 microM) stimulated significantly PIP2 hydrolysis, IP3 production, PA formation, and contraction. (2) Addition of ISO (0.1-25 microM), which raises the tissue cAMP level, to muscle precontracted with CCh attenuated PIP2 hydrolysis, IP3 production, PA formation and contraction in a time- and dose-dependent manner. (3) AlF4- (10 microM) induced a slow but progressive hydrolysis of PIP2, accompanied by parallel production of IP3, formation of PA, and contraction of the smooth muscle. The effects of AlF4- were dose-dependent and inhibited by deferoxamine, an Al3+ ion chelator. (4) Both forskolin (1-25 microM), which directly stimulates adenylate cyclase, and ISO inhibited the responses induced by AlF4- (10 microM) in a dose-dependent manner. (5) NaF (1-5 mM) had no effect on the activity of phospholipase C (PLC), purified from bovine iris sphincter. Furthermore, phosphorylation of the enzyme by catalytic subunit of protein kinase A had no inhibitory effect on PLC activity against PIP2. In conclusion, neither the muscarinic receptor nor PLC are the target sites for cAMP inhibition; instead the putative G-protein, which couples the activated muscarinic receptor to PLC, may be phosphorylated by cAMP-dependent protein kinase. This could attenuate the stimulation of PLC by the G-protein, thus resulting in inhibition of PIP2 hydrolysis and consequently leading to muscle relaxation. These results demonstrate cross-talk between the cAMP and IP3-Ca2+ second messenger systems and suggest that this could constitute a regulatory mechanism for the process of contraction-relaxation in smooth muscle.
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PMID:Effects of isoproterenol and forskolin on carbachol- and fluoroaluminate-induced polyphosphoinositide hydrolysis, inositol trisphosphate production, and contraction in bovine iris sphincter smooth muscle: interaction between cAMP and IP3 second messenger systems. 131 46


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