Science.gov

Sample records for adenylate cyclase response

  1. Ultraviolet radiation augments epidermal beta-adrenergic adenylate cyclase response

    SciTech Connect

    Iizuka, H.; Kajita, S.; Ohkawara, A.

    1985-05-01

    Pig skin was irradiated in vivo with fluorescent sunlamp tubes (peak emission at 305 nm). A significant increase in epidermal beta-adrenergic adenylate cyclase response was observed as early as 12 h following 1-2 minimum erythema doses (MEDs) UVB exposure, which lasted at least 48 h. The augmentation of adenylate cyclase response was relatively specific to the beta-adrenergic system and there was no significant difference in either adenosine- or histamine-adenylate cyclase response of epidermis. The increased beta-adrenergic adenylate cyclase response was less marked at higher doses of UVB exposure (5 MEDs); in the latter condition, a significant reduction in adenosine- or histamine-adenylate cyclase response was observed. There was no significant difference in either low- or high-Km cyclic AMP phosphodiesterase activity between control and UVB-treated skin at 1-2 MEDs. These data indicate that the epidermal adenylate cyclase responses are affected in vivo by UVB irradiation, which might be a significant regulatory mechanism of epidermal cyclic AMP systems.

  2. Adenylate cyclase of human articular chondrocytes. Responsiveness to prostaglandins and other hormones.

    PubMed Central

    Houston, J P; McGuire, M K; Meats, J E; Ebsworth, N M; Russell, R G; Crawford, A; Mac Neil, S

    1982-01-01

    Adenylate cyclase [ATP pyrophosphate lyase (cyclizing), EC 4.6.1.1] was shown to be present in cultured human articular chondrocytes. Optimal conditions of incubation time, protein and substrate concentrations and pH were determined in whole cell lysates. Maximal activity occurred at pH 8.5 with no decrease in activity up to pH 10.0. Adenylate cyclase activity of particulate membrane preparations was enhanced by the addition of crude cytosol preparations. The prostaglandins E1, E2, F1 alpha, F2 alpha, D2, B1, B2, A1 and A2, as well as adrenaline and isoprenaline, stimulated adenylate cyclase derived from either adult or foetal chondrocytes. No significant stimulation was observed in the presence of human calcitonin or glucagon. Bovine parathyroid hormone always significantly stimulated the adenylate cyclase derived from foetal chondrocytes, but not from adult chondrocytes. Preincubation of the chondrocytes in culture with indomethacin and with or without supernatant medium from cultured mononuclear cells increased the responsiveness of the adenylate cyclase to prostaglandin E1. PMID:7159397

  3. Restoration of adenylate cyclase responsiveness in murine myeloid leukemia permits inhibition of proliferation by hormone. Butyrate augments catalytic activity of adenylate cyclase.

    PubMed

    Inhorn, L; Fleming, J W; Klingberg, D; Gabig, T G; Boswell, H S

    1988-04-01

    Mechanisms of leukemic cell clonal dominance may include aberrations of transmembrane signaling. In particular, neoplastic transformation has been associated with reduced capacity for hormone-stimulated adenylate cyclase activity. In the present study, prostaglandin E, a hormonal activator of adenylate cyclase that has antiproliferative activity in myeloid cells, and cholera toxin, an adenylate cyclase agonist that functions at a postreceptor site by activating the adenylate cyclase stimulatory GTP-binding protein (Gs), were studied for antiproliferative activity in two murine myeloid cell lines. FDC-P1, an interleukin 3 (IL 3)-dependent myeloid cell line and a tumorigenic IL 3-independent subline, FI, were resistant to these antiproliferative agents. The in vitro ability of the "differentiation" agent, sodium butyrate, to reverse their resistance to adenylate cyclase agonists was studied. The antiproliferative action of butyrate involved augmentation of transmembrane adenylate cyclase activity. Increased adenylate cyclase catalyst activity was the primary alteration of this transmembrane signaling group leading to the functional inhibitory effects on leukemia cells, although alterations in regulatory G-proteins appear to play a secondary role.

  4. GSK3β Mediates Renal Response to Vasopressin by Modulating Adenylate Cyclase Activity

    PubMed Central

    Patel, Satish; Hao, ChuanMing; Woodgett, James; Harris, Raymond

    2010-01-01

    Glycogen synthase kinase 3β (GSK3β), a serine/threonine protein kinase, is a key target of drug discovery in several diseases, including diabetes and Alzheimer disease. Because lithium, a potent inhibitor of GSK3β, causes nephrogenic diabetes insipidus, GSK3β may play a crucial role in regulating water homeostasis. We developed renal collecting duct-specific GSK3β knockout mice to determine whether deletion of GSK3β affects arginine vasopressin-dependent renal water reabsorption. Although only mildly polyuric under normal conditions, knockout mice exhibited an impaired urinary concentrating ability in response to water deprivation or treatment with a vasopressin analogue. The knockout mice had reduced levels of mRNA, protein, and membrane localization of the vasopressin-responsive water channel aquaporin 2 compared with wild-type mice. The knockout mice also expressed lower levels of pS256-AQP2, a phosphorylated form crucial for membrane trafficking. Levels of cAMP, a major regulator of aquaporin 2 expression and trafficking, were also lower in the knockout mice. Both GSK3β gene deletion and pharmacologic inhibition of GSK3β reduced adenylate cyclase activity. In summary, GSK3β inactivation or deletion reduces aquaporin 2 expression by modulating adenylate cyclase activity and cAMP generation, thereby impairing responses to vasopressin in the renal collecting duct. PMID:20056751

  5. Regulation of brain adenylate cyclase by calmodulin

    SciTech Connect

    Harrison, J.K.

    1988-01-01

    This thesis examined the interaction between the Ca{sup 2+}-binding protein, calmodulin (CaM), and the cAMP synthesizing enzyme, adenylate cyclase. The regulation of guanyl nucleotide-dependent adenylate cyclase by CaM was examined in a particulate fraction from bovine striatum. CaM stimulated basal adenylate cyclase activity and enhanced the stimulation of the enzyme by GTP and dopamine (DA). The potentiation of GTP- and DA-stimulated adenylate cyclase activities by CaM was more sensitive to the concentration of CaM than was the stimulation of basal activity. A photoreactive CaM derivative was developed in order to probe the interactions between CaM and the adenylate cyclase components of bovine brain. Iodo-({sup 125}I)-CaM-diazopyruvamide ({sup 125}I-CAM-DAP) behaved like native CaM with respect to Ca{sup 2+}-enhanced mobility on sodium dodecyl sulfate-polyacrylamide gels and Ca{sup 2+}-dependent stimulation of adenylate cyclase. {sup 125}I-CaM-DAP cross-linked to CaM-binding proteins in a Ca{sup 2+}-dependent, concentration-dependent, and CaM-specific manner. Photolysis of {sup 125}I-CaM-DAP and forskolin-agarose purified CaM-sensitive adenylate cyclase produced an adduct with a molecular weight of 140,000.

  6. Calmodulin independence of human duodenal adenylate cyclase.

    PubMed Central

    Smith, J A; Griffin, M; Mireylees, S E; Long, R G

    1991-01-01

    The calmodulin and calcium dependence of human adenylate cyclase from the second part of the duodenum was assessed in washed particulate preparations of biopsy specimens by investigating (a) the concentration dependent effects of free [Ca2+] on enzyme activity, (b) the effects of exogenous calmodulin on enzyme activity in ethylene glycol bis (b-aminoethyl ether)N,N'-tetra-acetic acid (EGTA) washed particulate preparations, and (c) the effects of calmodulin antagonists on enzyme activity. Both basal (IC50 = 193.75 (57.5) nmol/l (mean (SEM)) and NaF stimulated (IC50 = 188.0 (44.0) nmol/l) adenylate cyclase activity was strongly inhibited by free [Ca2+] greater than 90 nmol/l. Free [Ca2+] less than 90 nmol/l had no effect on adenylate cyclase activity. NaF stimulated adenylate cyclase activity was inhibited by 50% at 2.5 mmol/l EGTA. This inhibition could not be reversed by free Ca2+. The addition of exogenous calmodulin to EGTA (5 mmol/l) washed particulate preparations failed to stimulate adenylate cyclase activity. Trifluoperazine and N-(8-aminohexyl)-5-IODO-1-naphthalene-sulphonamide (IODO 8) did not significantly inhibit basal and NaF stimulated adenylate cyclase activity when measured at concentrations of up to 100 mumol/l. These results suggest that human duodenal adenylate cyclase activity is calmodulin independent but is affected by changes in free [Ca2+]. PMID:1752461

  7. Effects of UVB irradiation on epidermal adenylate cyclase responses in vitro: its relation to sunburn cell formation.

    PubMed

    Iizuka, H; Ishida-Yamamoto, A; Kajita, S; Tsutsui, M; Ohkuma, N

    1988-01-01

    UVB irradiation augmented the beta-adrenergic adenylate cyclase response of pig skin epidermis in vitro. The effect was observed 2-4 h following the irradiation and lasted at least for 48 h. There was no significant difference in cyclic AMP phosphodiesterase activity between control and UVB-irradiated epidermis at lower irradiation dose (150 mJ/cm2), which is the dose of the most marked beta-adrenergic augmentation effect. The augmentation effect was specific to the beta-adrenergic system; adenosine and histamine adenylate cyclase responses were unchanged or decreased depending on the irradiation dose. Histologically, marked sunburn-cell formation was observed following the UVB irradiation. It has been suggested that oxygen intermediates generated by ultraviolet radiation participate in sunburn-cell formation. The addition of superoxide dismutase (SOD) in the incubation medium significantly inhibited sunburn-cell formation. On the other hand, the beta-adrenergic augmentation effect was not affected by the addition of SOD. Other scavengers of oxygen intermediates (catalase, catalase + SOD, xanthine, or mannitol) did not inhibit the UVB-induced beta-adrenergic augmentation effect. Further, superoxide-anion generating systems (hypoxanthine-xanthine oxidase system and acetaldehyde-xanthine oxidase system) revealed no stimulatory effect on the beta-adrenergic response of epidermis. These results indicate that (a) the UVB-induced beta-adrenergic augmentation effect is inherent to skin and does not depend on systemic factors such as inflammatory infiltrates following UVB irradiation; (b) in contrast to sunburn-cell formation, induction of the beta-adrenergic adenylate cyclase response is not directly associated with oxygen intermediates generated by UVB irradiation.

  8. WR-2721 inhibits parathyroid adenylate cyclase

    SciTech Connect

    Weaver, M.E.; Morrissey, J.; McConkey, C. Jr.; Goldfarb, S.; Slatopolsky, E.; Martin, K.J.

    1987-02-01

    WR-2721 (S-2-(3-aminopropylamino)ethylphosphorothioic acid) is a chemoprotective and radioprotective agent that has been shown to lower serum calcium in dogs and in humans. This is secondary both to impaired release of CaS from bone and diminished secretion of parathyroid hormone (PTH) from parathyroid glands. Because cAMP plays a role in the regulation of PTH secretion and WR-2721 has been shown to lower cAMP levels in radiated mouse spleen, the authors investigated the effects of WR-2721 on cAMP production in dispersed bovine parathyroid cells. Additional, they studied the adenylate cyclase in plasma membranes from normal bovine parathyroid glands after exposure to WR-2721. With parathyroid cells incubated at 0.5 mM CaS , addition of Wr-2721 in concentrations ranging from 0.02 to 2.0 mM resulted in a progressive decrease in intracellular cAMP measured by radioimmunoassay. In plasma membranes of bovine parathyroid cells a dose-dependent decrease in adenylate cyclase activity was noted. Inhibition of the cyclase was seen over a wide range of MgS concentrations. WR-2721 inhibited both basal and NaF, Gpp(NH)(, forskolin, and pertussin toxin-stimulated adenylate cyclase. These data suggest that WR-2721 inhibits the activity of parathyroid adenylate cyclase.

  9. Bordetella adenylate cyclase toxin: entry of bacterial adenylate cyclase into mammalian cells.

    PubMed

    Confer, D L; Slungaard, A S; Graf, E; Panter, S S; Eaton, J W

    1984-01-01

    We have identified an adenylate cyclase toxin in urea extracts and culture supernatant fluids of Bordetella pertussis (2). The ability of this toxin and the lack of a strong correlation between its activity and adenylate cyclase activity found in urea extracts suggest that it is an oligomer of readily dissociable subunits. The mechanism by which Bordetella adenylate cyclase toxin interacts with target cells is unknown, but polyvalent cations are necessary. Neutrophils exposed to the toxin acquire a 39,000 Mr protein that can also be photoaffinity labeled with 32P-ATP. We anticipate that this protein will prove to be a catalytic component of Bordetella adenylate cyclase toxin. Susceptible cells exposed to Bordetella adenylate cyclase toxin are functionally aberrant. In phagocytes, decreased bactericidal capacity may be important in the pathogenesis of human whooping cough and other Bordetella infections occurring in domestic animals. The effects of the toxin on neoplastic cells may offer new insights into the factors controlling their growth and differentiation. Bordetella adenylate cyclase toxin is a unique bacterial product. Further purification and characterization of this toxin will add to our understanding of cell-protein interactions and pathogen-host relationships.

  10. Glucose Inhibition of Adenylate Cyclase in Intact Cells of Escherichia coli B

    PubMed Central

    Peterkofsky, Alan; Gazdar, Celia

    1974-01-01

    Previous studies in E. coli B have demonstrated an inverse correlation between the presence of glucose in the medium and the accumulation of cyclic AMP in the medium. This observation could not be explained by the action of glucose as a repressor of adenylate cyclase (EC 4.6.1.1) synthesis, as a stabilizer of cyclic AMP phosphodiesterase (EC 3.1.4.17) activity, or as a direct inhibitor of adenylate cyclase activity in cell-free preparations. The recent development of an in vivo assay for adenylate cyclase has provided a basis for further exploring the inhibitory action of glucose in intact cells. With this assay it has been possible to show that, while glucose does not affect adenylate cyclase in vitro, it rapidly inhibits the enzyme activity in intact cells. Extensive metabolism of glucose is not required, since α-methylglucoside also inhibits adenylate cyclase in vivo. When cells are grown on glucose as carbon source, some sugars (mannose, glucosamine) substitute for glucose as adenylate cyclase inhibitors while others (e.g., fructose) do not. Dose-response studies indicate that low concentrations of glucose lead to essentially complete inhibition of adenylate cyclase activity while only moderately decreasing intracellular cyclic AMP concentrations. The evidence presented suggests that the decreased cellular cyclic AMP levels resulting from glucose addition can be accounted for by inhibition of adenylate cyclase without any significant effect on cyclic AMP phosphodiesterase or the transport of cyclic AMP from the cells to the medium. PMID:4366761

  11. Studies on responsiveness of hepatoma cells to catecholamines. III. Difference between the receptor-adenylate cyclase regulating systems in AH130 cells and cultured normal rat liver cells.

    PubMed

    Sanae, F; Matsunaga, T; Miyamoto, K; Koshiura, R

    1986-10-01

    The responsiveness to three beta-adrenergic agonists, isoproterenol (IPN), epinephrine (Epi) and norepinephrine (NE) in AH13O cells was examined compared with that in normal rat liver cells which were cultured for 24 hr after collagenase digestion. As regards to the activation of adenylate cyclase in the cell homogenates, the relative affinity of the three agonists was in order of IPN greater than NE greater than Epi in AH130 cells and IPN greater than Epi greater than NE in cultured normal liver cells. While the efficacies of the three agonists were similar in cultured liver cells, those of NE and Epi were markedly lower than that of IPN in AH13O cells and were increased to the similar level of IPN by pretreatment with phentolamine, but not with prazosin. Clonidine inhibited the activation of adenylate cyclase by IPN in AH13O cells. When cells were preincubated with islet-activating protein (IAP), the activity of adenylate cyclase in the presence or absence of agonist in both cell lines increased. In IAP-treated AH13O cells, the efficacies of NE and Epi became close to that of IPN. Adenylate cyclase in IAP-treated AH13O cells was activated by GTP in a dose-dependent manner, but that in IAP-treated cultured liver cells was not. In the presence of IPN, biphasic (activatory and inhibitory) effects of GTP on the cyclase were observed, and the inhibitory phase was eliminated by the IAP-treatment in both cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Role of Bordetella pertussis RseA in the cell envelope stress response and adenylate cyclase toxin release

    PubMed Central

    Hanawa, Tomoko; Yonezawa, Hideo; Kawakami, Hayato; Kamiya, Shigeru; Armstrong, Sandra K.

    2013-01-01

    Bordetella pertussis is the bacterial agent of the human disease, whooping cough. In many bacteria, the extracellular function sigma factor σE is central to the response to envelope stress, and its activity is negatively controlled by the RseA anti-sigma factor. In this study, the role of RseA in B. pertussis envelope stress responses was investigated. Compared with the wild-type strain, an rseA mutant showed elevated resistance to envelope stress and enhanced growth at 25°C. rpoH and other predicted σE target genes demonstrated increased transcription in the rseA mutant compared with the wild type parent. Transcription of those genes was also increased in wild type B. pertussis and Escherichia coli under envelope stress, whereas no stress-induced increase in transcription was observed in the rseA mutant. rseA inactivation was also associated with altered levels of certain proteins in culture supernatant fluids, which showed increased adenylate cyclase toxin (CyaA) levels. The increased CyaA in the mutant was correlated with an apparent increased stability of the extracellular toxin and increased production of CyaA-containing outer membrane vesicles. Consistent with this, compared with the wild type strain, rseA mutant cells produced increased numbers of large surface-associated vesicles. PMID:23821542

  13. Interaction of Trypanosoma cruzi adenylate cyclase with liver regulatory factors.

    PubMed Central

    Eisenschlos, C; Flawiá, M M; Torruella, M; Torres, H N

    1986-01-01

    Trypanosoma cruzi adenylate cyclase catalytic subunits may interact with regulatory factors from rat liver membranes, reconstituting heterologous systems which are catalytically active in assay mixtures containing MgATP. The systems show stimulatory responses to glucagon and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) or fluoride. Reconstitution was obtained by three different methods: fusion of rat liver membranes (pretreated with N-ethylmaleimide) to T. cruzi membranes; interaction of detergent extracts of rat liver membranes with T. cruzi membranes; or interaction of purified preparations of T. cruzi adenylate cyclase and of liver membrane factors in phospholipid vesicles. The liver factors responsible for the guanine nucleotide effect were characterized as the NS protein. Data also indicate that reconstitution requires the presence of a membrane substrate. PMID:2947568

  14. Forskolin activation of serotonin-stimulated adenylate cyclase in the liver fluke Fasciola hepatica.

    PubMed

    McNall, S J; Mansour, T E

    1985-05-15

    Properties of forskolin activation of adenylate cyclase in the liver fluke Fasciola hepatica are described. Forskolin stimulated adenylate cyclase activity in cell-free fluke particles to levels more than 30-fold above the basal rate. This activation was not dependent on guanine nucleotides and, upon washing of the particles, was rapidly reversed. Forskolin potentiated the activation of adenylate cyclase by serotonin (5-HT) and lysergic acid diethylamide (LSD), resulting in both an increase in the maximal level of enzyme activity and a decrease in the apparent activation constant (KA). The 5-HT antagonist 2-bromo-LSD did not inhibit enzyme activation by forskolin. Furthermore, forskolin had no effect on specific [3H]LSD binding to fluke particles. Activation of adenylate cyclase by sodium fluoride or guanine nucleotides was modified in a complex manner by forskolin with both stimulatory and inhibitory effects present. The results suggest that forskolin does not interact directly with the 5-HT receptor coupled to adenylate cyclase. Instead, it appears that forskolin effects are, at least in part, due to its ability to alter the interaction between the regulatory and catalytic components of adenylate cyclase. Incubation of intact flukes with forskolin increased their cAMP levels 2- to 3-fold. The concentration dependence of this response was similar to that for forskolin activation of adenylate cyclase in fluke particles, with 300 microM forskolin giving the maximum response. Forskolin and other agents that increased fluke cAMP levels also stimulated fluke motility.

  15. Antineoplastic effects of Bordetella pertussis adenylate cyclase.

    PubMed

    Slungaard, A; Confer, D L; Jacob, H S; Eaton, J W

    1983-01-01

    Urea extracts of B. pertussis, but not B. bronchiseptica, cause large and sustained intracellular cAMP elevation in several neoplastic cell lines. These cAMP elevations are associated with growth inhibition (HL-60, Friend erythroleukemia) and a phenotypic change/differentiation (HL-60, L1210). B. pertussis extract injections prolong survival of L1210 tumor-bearing mice. Pretreatment of L1210 cells with B. pertussis extract both delays mortality and induces growth of solid tumors instead of ascites in subsequently inoculated mice. We conclude that B. pertussis adenylate cyclase is capable of invading a variety of neoplastic cells to catalyze the intracellular formation of large amounts of cAMP. These cAMP elevations are durable and promote growth arrest, differentiation, or phenotypic alterations reflected in altered biologic behavior. B. pertussis adenylate cyclase should prove to be a useful tool for manipulating cAMP levels in neoplastic cells to elucidate the role of cAMP in malignant transformation.

  16. Cellular interactions uncouple beta-adrenergic receptors from adenylate cyclase.

    PubMed

    Ciment, G; de Vellis, J

    1978-11-17

    C6 glioma cells and B104 neuroblastoma cells both possess adenylate cyclase activity, but only C6 cells have beta-adrenergic receptors. However, when cocultured with B104 cells, C6 cells show a marked decrease in their ability to accumulate adenosine 3', 5'-monophosphate upon stimulation with beta receptor agonists. Since both beta receptors and cholera toxin-stimulated adenylate cyclase activities are present in C6/B104 cocultures, we conclude that the beta receptor/adenylate cyclase transduction mechanism in cocultured C6 cells is uncoupled.

  17. Reduced early and late phase insulin response to glucose in isolated spiny mouse (Acomys cahirinus) islets: a defective link between glycolysis and adenylate cyclase.

    PubMed

    Nesher, R; Abramovitch, E; Cerasi, E

    1989-09-01

    The spiny mouse (Acomys cahirinus) exhibits low insulin responsiveness to glucose with a nearly absent early phase release. The alternative fuel-secretagogue glyceraldehyde (10 mmol/l) produced a maximal early insulin response in rat islets but failed to affect early response in Acomys; however, it potentiated the late insulin response in both species alike. Glucagon (1.5 mumol/l) potentiated the early insulin response to intermediate (8.3 mmol/l) glucose in rat and Acomys islets by two- and four-fold, respectively. Glucose doubled cyclic AMP levels in rat islets but no significant response was noted in Acomys islets. Isobutylmethylxanthine (0.1 mmol/l) and forskolin (25 mumol/l) caused a significant rise in islet cyclic AMP levels in both types of islets; however, neither agent restored the glucose stimulation of cyclic AMP in spiny mouse islets. Forskolin and isobutylmethylxanthine potentiated early and late phase insulin release in both species; however, neither augmented the early response in the Acomys to the degree observed in rat islets. Thus: (1) A deficient link exists in Acomys between glycolysis and subsequent signals. (2) These islets contain a glucose-insensitive adenylate cyclase. (3) The early insulin response may be potentiated by direct activation of adenylate cyclase. (4) The glucose effects on early and late phase insulin release are probably mediated by distinct pathways. (5) In the spiny mouse the signals mediating the early response are deranged to a greater extent than those activating the late phase insulin release.

  18. Suppression of the humoral immune response by cannabinoids is partially mediated through inhibition of adenylate cyclase by a pertussis toxin-sensitive G-protein coupled mechanism.

    PubMed

    Kaminski, N E; Koh, W S; Yang, K H; Lee, M; Kessler, F K

    1994-11-16

    Cannabinoid compounds, including the major psychoactive component of marihuana, delta 9-tetrahydrocannabinol (delta 9-THC), have been widely established as being inhibitory on a broad array of humoral and cell-mediated immune responses. The presence of cannabinoid receptors has been identified recently on mouse spleen cells, which possess structural and functional characteristics similar to those of the G-protein coupled cannabinoid receptor originally identified in rat brain. These findings, together with those demonstrating that delta 9-THC inhibits adenylate cyclase in splenocytes, strongly suggest that certain aspects of immune inhibition by cannabinoids may be mediated through a cannabinoid receptor-associated mechanism. The objective of the present studies was to determine whether inhibition of adenylate cyclase is relevant to mouse spleen cell immune function and, if so, whether this inhibition is mediated through a Gi-protein coupled mechanism as previously described in neuronal tissue. Spleen cell activation by the phorbol ester phorbol-12-myristate-13-acetate (PMA), plus the calcium ionophore ionomycin, produced a rapid but transient increase in cytosolic cAMP, which was inhibited completely by immunosuppressive concentrations of delta 9-THC (22 microM) and the synthetic bicyclic cannabinoid CP-55940 (5.2 microM), which produced no effect on cell viability. Inhibition by cannabinoids of lymphocyte proliferative responses to PMA plus ionomycin and sheep erythrocyte (sRBC) IgM antibody-forming cell (AFC) response, was abrogated completely by low concentrations of dibutyryl-cAMP (10-100 microM). Inhibition of the sRBC AFC response by both delta 9-THC (22 microM) and CP-55940 (5.2 microM) was also abrogated by preincubation of splenocytes for 24 hr with pertussis toxin (0.1-100 ng/mL). Pertussis toxin pretreatment of spleen cells was also found to directly abrogate cannabinoid inhibition of adenylate cyclase, as measured by forskolin-stimulated accumulation

  19. Phorbol esters alter adenylate cyclase responses to vasoactive intestinal peptide and forskolin in the GH cell line

    SciTech Connect

    Summers, S.; Florio, T.; Cronin, M.

    1986-05-01

    Activation of protein kinase C with phorbol ester modifies cyclic AMP production in several anterior pituitary cell systems. In the GH cell line from a rat pituitary tumor, exposure to phorbol 12-myristate 13-acetate (PMA: 100 nM) for 30 minutes significantly reduces vasoactive intestinal peptide (VIP: 100 nM) stimulated adenylate cyclase (AC) activity in subsequent membrane preparations to 62 + 4% of control (n = 6 independent studies). In contrast, these same membrane preparations respond to forskolin (1 ..mu..M) with significantly more activity, 130 +/- 6% of controls (n = 6 independent studies). Finally, phorbol ester does not block an inhibitory hormone input into the AC system; somatostatin (100 nM) reduction of VIP-stimulated AC activity is not significantly different in membrane preparations from PMA treated and control cells (n = 3 independent studies). These other findings lead the authors to propose that protein kinase C can modify several sites in the AC complex in anterior pituitary cells.

  20. Adenylate cyclase activity in a higher plant, alfalfa (Medicago sativa).

    PubMed Central

    Carricarte, V C; Bianchini, G M; Muschietti, J P; Téllez-Iñón, M T; Perticari, A; Torres, N; Flawiá, M M

    1988-01-01

    An adenylate cyclase activity in Medicago sativa L. (alfalfa) roots was partially characterized. The enzyme activity remains in the supernatant fluid after centrifugation at 105,000 g and shows in crude extracts an apparent Mr of about 84,000. The enzyme is active with Mg2+ and Ca2+ as bivalent cations, and is inhibited by EGTA and by chlorpromazine. Calmodulin from bovine brain or spinach leaves activates this adenylate cyclase. PMID:3128270

  1. Developmental changes of beta-adrenergic receptor-linked adenylate cyclase of rat liver

    SciTech Connect

    Katz, M.S.; Boland, S.R.; Schmidt, S.J.

    1985-06-01

    beta-Adrenergic agonist-sensitive adenylate cyclase activity and binding of the beta-adrenergic antagonist(-)-(/sup 125/I)iodopindolol were studied in rat liver during development of male Fischer 344 rats ages 6-60 days. In liver homogenates maximum adenylate cyclase response to beta-adrenergic agonist (10(-5) M isoproterenol or epinephrine) decreased by 73% (P less than 0.01) between 6 and 60 days, with most of the decrease (56%; P less than 0.01) occurring by 20 days. beta-adrenergic receptor density (Bmax) showed a corresponding decrease of 66% (P less than 0.01) by 20 days without subsequent change. Binding characteristics of stereospecificity, pharmacological specificity, saturability with time, and reversibility were unchanged with age. GTP-, fluoride-, forskolin-, and Mn2+-stimulated adenylate cyclase activities also decreased during development, suggesting a decrease of activity of the catalytic component and/or guanine nucleotide regulatory component of adenylate cyclase. These results indicate that the developmental decrease of beta-adrenergic agonist-sensitive adenylate cyclase activity may result from decreased numbers of beta-adrenergic receptors. Developmental alterations of nonreceptor components of the enzyme may also contribute to changes of catecholamine-sensitive adenylate cyclase.

  2. Studies on responsiveness of hepatoma cells to catecholamines. VI. Characteristics of adrenoceptors and adenylate cyclase response in rat ascites hepatoma cells and human hepatoma cells.

    PubMed

    Sanae, F; Kohei, K; Nomura, M; Miyamoto, K

    1992-06-01

    Alpha 1, alpha 2- and beta-Adrenoceptor densities and catecholamine responsiveness in established hepatoma cells, rat ascites hepatoma AH13, AH66, AH66F, AH109A, AH130 and AH7974 cells and human hepatocellular carcinoma HLF and HepG2 cells, were compared with those in normal rat hepatocytes and Chang liver cells. Alpha 1-Adrenoceptor densities measured by [3H]prazosin bindings were not detected in all hepatoma cell lines. Alpha 2-Adrenoceptor densities measured by [3H]clonidine bindings were also barely detected in hepatoma cell lines except for AH130 cells and HepG2 cells. Regarding beta-adrenoceptor, AH109A, AH130 and AH7974 cells had much more [125I]iodocyanopindolol binding sites than normal rat hepatocytes, although we could not detect the binding in HepG2 cells. Adenylate cyclase of normal rat hepatocyte and Chang liver cells were stimulated by beta 2-adrenergic agonist salbutamol, while the cyclase in hepatoma cells had no beta 2-adrenergic response but a beta 1-type response. These findings indicate that the characteristics of adrenergic response in hepatoma cell lines is very different from that in normal hepatocytes, suggesting a participation in the hepatocarcinogenesis and/or the autonomous proliferation of hepatoma cells.

  3. Activation of fat cell adenylate cyclase by protein kinase C

    SciTech Connect

    Naghshineh, S.; Noguchi, M.; Huang, K.P.; Londos, C.

    1986-05-01

    Purified protein kinase C (C-kinase) from guinea pig pancreas and rat brain stimulated adenylate cyclase activity in purified rat adipocyte membranes. Cyclase stimulation occurred over 100 to 1000 mU/ml of C-kinase activity, required greater than 10 ..mu..M calcium, proceeded without a lag, was not readily reversible, and required no exogenous phospholipid. Moreover, C-kinase inhibitors, such as chlorpromazine and palmitoyl carnitine, inhibited selectively adenylate cyclase which was activated by C-kinase and calcium. Depending on assay conditions, 10 nM 12-0-tetradecanoylphorbol-13-acetate (TPA) either enhanced or was required for kinase action on cyclase. Also, TPA plus calcium promoted the quantitative association of C-kinase with membranes. Adenylate cyclase activation by C-kinase was seen both in the presence and absence of exogenous GTP, indicating that the kinase effect does not result from an action on the GTP-binding, inhibitory regulatory component (N/sub i/) of the cyclase system. Moreover, the kinase effect was seen in the presence of non-phosphorylating ATP analogs, such as AppNHp and AppCH/sub 2/p, suggesting that the effects of C-kinase described herein may result from association with, rather than phosphorylation of, adenylate cyclase.

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

    SciTech Connect

    Cronin, M.J.; Evans, W.S.; Rogol, A.D.; Weiss, A.A.; Thorner, M.O.; Orth, D.N.; Nicholson, W.E.; Yasumoto, T.; Hewlett, E.L.

    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 amplified 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.

  5. Interaction of 7-bromoacetyl-7-desacetylforskolin with adenylate cyclase

    SciTech Connect

    Laurenza, A.; Morris, D.I.; Seamon, K.B.

    1986-05-01

    7-Bromoacetyl-7-desacetylforskolin (BrAcFk) and the 12-tritio derivative (/sup 3/H-BrAckFk) were synthesized as alkylating analogs of forskolin. BrAcFk stimulated adenylate cyclase in human platelet and bovine brain membranes with an EC50 of 50..mu..M and inhibited /sup 3/H-forskolin binding to these membranes with a K/sub i/ of 300 nM. /sup 3/H-forskolin binding was decreased in membranes pretreated for 20 min with 10 ..mu..M BrAcFk. The i,9-dideoxy derivative of BrAcFk did not activate adenylate cyclase or inhibit /sup 3/H-forskolin binding. Proteins labelled by BrAcFk in solubilized preparations from bovine brain and human platelets were identified by fluorography of SDS gels. The two predominant bands labelled in the low and high molecular weight regions had molecular weights of 50,000 and 135,000 daltons respectively. The 135,000 dalton band identified by fluorography coeluted with adenylate cyclase activity on a Dupont GF450 column and has a molecular weight identical to that of the catalytic subunit determined by silver staining of SDS gels. These results suggest that BrAcFk can react covalently with the catalytic subunit of adenylate cyclase.

  6. Virulence of Bordetella bronchiseptica: role of adenylate cyclase-hemolysin.

    PubMed Central

    Gueirard, P; Guiso, N

    1993-01-01

    Bordetella bronchiseptica is a pathogen of laboratory, domestic, and wild animals and sometimes of humans. In the present study some characteristics of the virulence of B. bronchiseptica isolates of different origin were studied. All isolates had similar phenotypes, similar bacteriological characters, and synthesized adenylate cyclase-hemolysin, filamentous hemagglutinin and pertactin but not pertussis toxin. These isolates, however, differed in their ability to express dermonecrotic toxin and to cause a lethal infection, but no correlation was found with the human or animal origin of the isolates. The fact that the most virulent isolate did not express dermonecrotic toxin suggests that this toxin does not play an important role in the virulence of the bacteria in the murine model. After infection with virulent B. bronchiseptica a very early synthesis and a persistence of anti-adenylate cyclase-hemolysin and anti-filamentous hemagglutinin antibodies were observed in the sera of infected mice, suggesting a persistence of the bacteria or of its antigens. B. bronchiseptica adenylate cyclase-hemolysin was purified and was shown to be a major protective antigen against B. bronchiseptica infection. Furthermore, we showed that its immunological and protective properties were different from that of B. pertussis adenylate cyclase-hemolysin, confirming that Bordetella species are immunologically different. Images PMID:8406794

  7. Modification of adenylate cyclase by photoaffinity analogs of forskolin

    SciTech Connect

    Ho, L.T.; Nie, Z.M.; Mende, T.J.; Richardson, S.; Chavan, A.; Kolaczkowska, E.; Watt, D.S.; Haley, B.E.; Ho, R.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 following 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.

  8. Engineering adenylate cyclases regulated by near-infrared window light.

    PubMed

    Ryu, Min-Hyung; Kang, In-Hye; Nelson, Mathew D; Jensen, Tricia M; Lyuksyutova, Anna I; Siltberg-Liberles, Jessica; Raizen, David M; Gomelsky, Mark

    2014-07-15

    Bacteriophytochromes sense light in the near-infrared window, the spectral region where absorption by mammalian tissues is minimal, and their chromophore, biliverdin IXα, is naturally present in animal cells. These properties make bacteriophytochromes particularly attractive for optogenetic applications. However, the lack of understanding of how light-induced conformational changes control output activities has hindered engineering of bacteriophytochrome-based optogenetic tools. Many bacteriophytochromes function as homodimeric enzymes, in which light-induced conformational changes are transferred via α-helical linkers to the rigid output domains. We hypothesized that heterologous output domains requiring homodimerization can be fused to the photosensory modules of bacteriophytochromes to generate light-activated fusions. Here, we tested this hypothesis by engineering adenylate cyclases regulated by light in the near-infrared spectral window using the photosensory module of the Rhodobacter sphaeroides bacteriophytochrome BphG1 and the adenylate cyclase domain from Nostoc sp. CyaB1. We engineered several light-activated fusion proteins that differed from each other by approximately one or two α-helical turns, suggesting that positioning of the output domains in the same phase of the helix is important for light-dependent activity. Extensive mutagenesis of one of these fusions resulted in an adenylate cyclase with a sixfold photodynamic range. Additional mutagenesis produced an enzyme with a more stable photoactivated state. When expressed in cholinergic neurons in Caenorhabditis elegans, the engineered adenylate cyclase affected worm behavior in a light-dependent manner. The insights derived from this study can be applied to the engineering of other homodimeric bacteriophytochromes, which will further expand the optogenetic toolset.

  9. 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.

  10. Mechanisms of nonhormonal activation of adenylate cyclase based on target analysis

    SciTech Connect

    Verkman, A.S.; Ausiello, D.A.; Jung, C.Y.; Skorecki, K.L.

    1986-08-12

    Radiation inactivation was used to examine the mechanism of activation of adenylate cyclase in the cultured renal epithelial cell line LLC-PK1 with hormonal (vasopressin) and nonhormonal (GTP, forskolin, fluoride, and chloride) activating ligands. Intact cells were frozen, irradiated at -70 degrees C (0-14 Mrad), thawed, and assayed for adenylate cyclase activity in the presence of activating ligands. The ln (adenylate cyclase activity) vs. radiation dose relation was linear (target size 162 kDa) for vasopressin- (2 microM) stimulated activity and concave downward for unstimulated (10 mM Mn/sup 2 +/), NaF- (10 mM) stimulated, and NaCl- (100 mM) stimulated activities. Addition of 2 microM vasopressin did not alter the ln activity vs. dose relation for NaF- (10 mM) stimulated activity. The dose-response relations for adenylate cyclase activation and for transition in the ln activity vs. dose curve shape were measured for vasopressin and NaF. On the basis of our model for adenylate cyclase subunit interactions reported previously (Verkman, A. S., Skorecki, K. L., and Ausiello, D. A. (1986) Am. J. Physiol. 260, C103-C123) and of new mathematical analyses, activation mechanisms for each ligand are proposed. In the unstimulated state, equilibrium between alpha beta and alpha + beta favors alpha beta; dissociated alpha binds to GTP (rate-limiting step), which then combines with the catalytic (C) subunit to form active enzyme. Vasopressin binding to receptor provides a rapid pathway for GTP binding to alpha. GTP and its analogues accelerate the rate of alpha GTP formation. Forskolin inhibits the spontaneous deactivation of activated C. Activation by fluoride may occur without alpha beta dissociation or GTP addition through activation of C by an alpha beta-F complex.

  11. [Adenylate cyclase. A possible factor in the pathogenicity of Yersinia pestis].

    PubMed

    Michankin, B N; Chevchenko, L A; Asseeva, L E

    1992-01-01

    Biological effect of homogenous preparation of Y. pestis adenylate cyclase on eucaryotic cells was studied. Adenylate cyclase, added (7.5 x 10(8) g/ml) to guinea pig macrophages lowers the level of chemiluminescence to 50-70%, has an appreciable cytotoxic effect on peritoneal macrophages and suppresses phosphorylation processes of leucocyte proteins from white mice. The experimental results obtained allow to suggest Y. pestis adenylate cyclase to be a pathogenic factor, contributing to the development of plague infection.

  12. Adenylate cyclase and the search for new compounds with the clinical profile of lithium.

    PubMed

    Belmaker, R H

    1984-01-01

    It is possible to evaluate the beta-adrenergic receptor-adenylate cyclase complex in the human periphery by measuring the plasma cyclic AMP rise after adrenergic agonists. A clinical trial of the beta 2 adrenergic agonist salbutamol in depression provided an opportunity to test whether adrenergic receptor subsensitivity does occur during clinical antidepressant treatment. After 1 and 3 weeks of oral salbutamol treatment, depression scores declined significantly in 11 depressed patients, while the plasma cyclic AMP response to i.v. salbutamol declined over 60%. The results support the concept that receptor sensitivity changes occur during human antidepressant therapy. Data are presented that Li, too, markedly reduces activity of beta-adrenergic adenylate cyclase in humans. The effect was evaluated by studying the effect of Li at therapeutic serum concentrations on the plasma cyclic AMP response to subcutaneous epinephrine. The Li effect is specific, since the plasma cyclic AMP response to glucagon is not inhibited. In rat cortical slices Li inhibition of noradrenaline-induced cyclic AMP accumulation is clearly demonstrable only at concentrations close to 2 mM Li. However, fresh human brain slices from edges of surgically-removed tumors show Li inhibition at 1 mM Li concentrations. These results imply that in brain as well as periphery, human noradrenergic adenylate cyclase is inhibited by therapeutic concentrations of Li. Demeclocyclin, a tetracycline-derived antibiotic, was found to inhibit noradrenaline-sensitive adenylate cyclase in rat cortical slices and to inhibit amphetamine-induced hyperactivity in rats in an open field. Clinical trials should search for new compounds with the clinical profile of Li.

  13. Multiple effects of phorbol esters on hormone-sensitive adenylate cyclase activity in S49 lymphoma cells

    SciTech Connect

    Bell, J.D.; Brunton, L.L.

    1987-06-01

    In S49 lymphoma cells, 12-O-tetradecanoyl phorbol-13-acetate (TPA) enhances adenylate cyclase activity and doubles cAMP accumulation in response to ..beta..-adrenergic stimulation at 37/sup 0/C, putatively via the action of protein kinase C. at 27/sup 0/C, TPA has the opposite effect, inhibiting cAMP production in response to isoproterenol by approx. 25%. TPA also inhibits the response to prostaglandin E/sub 1/ (PGE/sub 1/), another stimulant of hormone-sensitive adenylate cyclase in these cells, by 30% at 37/sup 0/C and almost 50% at 27/sup 0/C. In contrast, TPA enhances responses to forskolin and cholera toxin at both 27 and 37/sup 0/C. In membranes from cells treated with TPA, PGE/sub 1/-stimulated adenylate cyclase activity is inhibited by 50%, whereas the catalytic activity stimulated by NaF or forskolin is enhanced. TPA reduces the potency of both PGE/sub 1/ and isoproterenol for cAMP generation by 50%. TPA causes a similar decrease in ..beta..-adrenergic agonist affinity with no reduction in the density of either antagonist of agonist binding sites in wild type cells and in cells lacking the ..cap alpha..-subunit of the stimulatory transducer protein (G/sub s/) (cyc/sup -/) or lacking functional receptor G/sub s/ coupling (UNC). Therefore, TPA has at least three functionally distinct effects on hormone-sensitive adenylate cyclase in S49 cells. The authors conclude that multiple and opposing effects of TPA on hormone-sensitive adenylate cyclase occur simultaneously within the same cell, affecting the responses to several agonists differently. In addition, the data offer a mechanism by which a cell can achieve heterogeneous efficacies to hormones that activate adenylate cyclase.

  14. Adenylate Cyclase Activity Not Found in Soybean Hypocotyl and Onion Meristem 1

    PubMed Central

    Yunghans, Wayne N.; Morré, D. James

    1977-01-01

    Tissue, homogenates, and purified cell fractions prepared from hypocotyls of a dicot, soybean (Glycine max), and meristematic tissue of a monocot, onion (Allium cepa), were examined critically for evidence of adenylate cyclase activity. Three assay methods were used: chemical analysis, isotope dilution analysis, and enzyme cytochemistry. In both crude extracts or whole tissue, as well as purified membranes, with or without auxin, no adenylate cyclase was detected by any of the three methods. For plasma membranes, the specific activity was less than 1/40 or 1/25,000 that of rat liver plasma membranes, depending on the assay procedure, i.e. below the limits of detection. Using comparable methods, we could detect neither cyclic adenosine 3′:5′-monophosphate nor the phosphodiesterase responsible for its degradation in either purified membranes or homogenates. The results suggest that hormone responses in plants are not generally mediated by a mechanism involving the obligate production of cyclic adenosine 3′:5′-monophosphate by a plasma membrane associated adenylate cyclase. Images PMID:16660026

  15. A Simple Luminescent Adenylate-Cyclase Functional Assay for Evaluation of Bacillus anthracis Edema Factor Activity

    PubMed Central

    Israeli, Ma’ayan; Rotem, Shahar; Elia, Uri; Bar-Haim, Erez; Cohen, Ofer; Chitlaru, Theodor

    2016-01-01

    Edema Factor (EF), the toxic sub-unit of the Bacillus anthracis Edema Toxin (ET) is a calmodulin-dependent adenylate cyclase whose detrimental activity in the infected host results in severe edema. EF is therefore a major virulence factor of B. anthracis. We describe a simple, rapid and reliable functional adenylate-cyclase assay based on inhibition of a luciferase-mediated luminescence reaction. The assay exploits the efficient adenylate cyclase-mediated depletion of adenosine tri-phosphate (ATP), and the strict dependence on ATP of the light-emitting luciferase-catalyzed luciferin-conversion to oxyluciferin, which can be easily visualized. The assay exhibits a robust EF-dose response decrease in luminescence, which may be specifically reverted by anti-EF antibodies. The application of the assay is exemplified in: (a) determining the presence of EF in B. anthracis cultures, or its absence in cultures of EF-defective strains; (b) evaluating the anti-EF humoral response in experimental animals infected/vaccinated with B. anthracis; and (c) rapid discrimination between EF producing and non-producing bacterial colonies. Furthermore, the assay may be amenable with high-throughput screening for EF inhibitory molecules. PMID:27548219

  16. Stimulatory and inhibitory effects of forskolin on adenylate cyclase in rat normal hepatocytes and hepatoma cells.

    PubMed

    Miyamoto, K; Sanae, F; Koshiura, R; Matsunaga, T; Takagi, K; Satake, T; Hasegawa, T

    1989-02-01

    Forskolin synergistically potentiated adenosine 3',5'-cyclic monophosphate formation by prostaglandin E1 (PGE1) in rat normal hepatocytes freshly prepared by collagenase digestion and rat ascites hepatoma AH66 cells, but dose-dependently inhibited the accumulation by PGE1 in AH66F cells. Forskolin activated adenylate cyclase in a dose-dependent manner in homogenates of all cell lines. In normal hepatocytes and AH66 cells, simultaneous addition of forskolin and other adenylate cyclase activators [isoproterenol (IPN), PGE1, guanosine 5'-triphosphate sodium salt (GTP), 5'-guanylylimidodiphosphate sodium salt (Gpp (NH)p), NaF, cholera toxin, islet activating protein and MnCl2] gave greater than additive responses. On the other hand, in AH66F cells, the effect of forskolin on adenylate cyclase was hardly influenced by GTP, but forskolin diminished the activities induced by high concentrations of GTP to that by the diterpene alone. Forskolin also significantly inhibited the PGE1-stimulated and the guanine nucleotide binding regulatory protein-stimulated activities. Because AH66F cells were insensitive to IPN, the combination with forskolin and IPN gave similar activity to that obtained with the diterpene alone. The effect of forskolin on the activation by manganese ion was neither synergistic nor inhibitory but was additive in AH66F cells. These results suggest that forskolin promotes the interaction between the stimulatory guanine nucleotide binding regulatory protein and the catalytic unit in normal hepatocytes and AH66 cells, but in AH66F cells forskolin interferes with the coupling of the two components of adenylate cyclase.

  17. Cloned M1 muscarinic receptors mediate both adenylate cyclase inhibition and phosphoinositide turnover.

    PubMed Central

    Stein, R; Pinkas-Kramarski, R; Sokolovsky, M

    1988-01-01

    The rat M1 muscarinic receptor gene was cloned and expressed in a rat cell line lacking endogenous muscarinic receptors. Assignment of the cloned receptors to the M1 class was pharmacologically confirmed by their high affinity for the M1-selective muscarinic antagonist pirenzepine and low affinity for the M2-selective antagonist AF-DX-116. Guanylyl imidodiphosphate [Gpp(NH)p] converted agonist binding sites on the receptor, from high-affinity to the low-affinity state, thus indicating that the cloned receptors couple to endogenous G-proteins. The cloned receptors mediated both adenylate cyclase inhibition and phosphoinositide hydrolysis, but by different mechanisms. Pertussis toxin blocked the inhibition of adenylate cyclase (indicating coupling of the receptor to inhibitory G-protein), but did not affect phosphoinositide turnover. Furthermore, the stimulation of phosphoinositide hydrolysis was less efficient than the inhibition of adenylate cyclase. These findings demonstrate that cloned M1 receptors are capable of mediating multiple responses in the cell by coupling to different effectors, possibly to different G-proteins. Images PMID:2846274

  18. Role of protein kinase C on the acute desensitization of renal cortical adenylate cyclase to parathyroid hormone.

    PubMed

    Bellorin-Font, E; López, C; Díaz, K; Pernalete, N; López, M; Starosta, R

    1995-01-01

    The mechanisms of adenylate cyclase desensitization to parathyroid hormone are still unclear. Current evidence suggest that the signal generated after PTH binding to receptors results in activation of adenylate cyclase and stimulation of phospholipase C with subsequent activation of protein kinase C. Recent studies have suggested a role of protein kinase C on the regulation of the PTH-dependent receptor-adenylate cyclase system in cultured cells. Therefore, the present studies were conducted to examine the role of protein kinase C on the desensitization of canine renal cortical adenylate cyclase after an acute exposure in vivo to PTH. A group of normal dogs were treated with a single intravenous injection of 1 microgram/k of syn bPTH (1-34) or Nle bPTH (3-34). Ten minutes later, animals were subjected to bilateral nephrectomy and the kidney cortex processed for preparations of basolateral membranes for determinations of adenylate cyclase activity, as well as membrane and cytosolic fractions for analysis of protein kinase C activity. Animals not treated with PTH were used as controls. PTH administration in vivo resulted in a 46.9 +/- 9.3% decrease in maximal adenylate cyclase activity in vitro in response to syn bPTH (1-34) (P < 0.001). Likewise, PTH binding as measured with 125I-Nle8,18,Tyr34-bPTH (1-34)NH2 showed a 40 +/- 3% decrease. This alterations were associated with a marked translocation of protein kinase C from the cytosol to the membrane. Thus, protein kinase C activity in membrane fractions increased from 160.6 +/- 44.8 pmol Pi/min in controls to 500.4 +/- 123 in PTH treated dogs (P < 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Microscopical localization on adenylate cyclase: a historical review of methodologies.

    PubMed

    Richards, P A; Richards, P D

    1998-03-15

    The histochemistry technique for localizing adenylate cyclase has been developed over the past two decades. Early efforts were directed at overcoming the criticism of the lead capture technique, the inhibition of the enzyme by fixation, and problems associated with the substrate. The introduction of alternative metal ions, strontium and cerium, offered solutions to the criticism of the lead capture technique. The inhibition of the enzyme by the various fixation methods used has been rarely overcome satisfactorily and the use of non-fixed material during incubation is one of the alternatives that has been suggested. The introduction of adenylate (beta-gamma-methylene) diphosphate as an alternative substrate offers a solution to the problems associated with commercially available adenylyl imidodiphosphate. Although no standard medium or method has been accepted by all researchers, the histochemical technique still has a place in the arsenal of the modern cell biologist. The technique localizes the active enzyme, as opposed to the protein, active and nonactive, by immunocytochemistry and the precursors of the protein by in situ hybridization methods.

  20. Dopaminergic modulation of adenylate cyclase stimulation by vasoactive intestinal peptide in anterior pituitary.

    PubMed Central

    Onali, P; Schwartz, J P; Costa, E

    1981-01-01

    The activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by vasoactive intestinal peptide (VIP) was used as a model to investigate the molecular mechanisms triggered by the occupancy of dopamine recognition sites in rat anterior pituitary. Dopamine failed to change the basal enzyme activity, but it inhibited the stimulation of adenylate cyclase elicited by VIP. Apomorphine, 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene, and 2-bromo-alpha-ergocryptine mimicked the effect of dopamine, whereas (-)-sulpiride and and classical neuroleptics antagonized it. Dopamine failed to modulate the activation of pituitary adenylate cyclase by prostaglandin E1, which does not increase prolactin secretion. From these results we infer that stimulation of D-2 (dopamine) receptors may affect pituitary secretion by inhibiting the activation of anterior pituitary adenylate cyclase by VIP or other secretagogues. PMID:6171819

  1. Evidence for adenylate cyclase as a scaffold protein for Ras2-Ira interaction in Saccharomyces cerevisie.

    PubMed

    Colombo, Sonia; Paiardi, Chiara; Pardons, Katrien; Winderickx, Joris; Martegani, Enzo

    2014-05-01

    Data in literature suggest that budding yeast adenylate cyclase forms a membrane-associated complex with the upstream components of the cAMP/PKA pathway. Here we provide evidences that adenylate cyclase (Cyr1p) acts as a scaffold protein keeping Ras2 available for its regulatory factors. We show that in a strain with deletion of the CYR1 gene (cyr1Δ pde2Δ msn2Δ msn4Δ) the basal Ras2-GTP level is very high and this is independent on the lack of feedback inhibition that could result from the absence of adenylate cyclase activity. Moreover, strains effected either in the intrinsic adenylate cyclase activity (fil1 strain) or in the stimulation of adenylate cyclase activity by active G-proteins (lcr1 strain) had a normal basal and glucose-induced Ras2-GTP level, indicating that adenylate cyclase activity does not influence the Ras2 activation state and suggesting that Cyr1 protein is required for the proper interaction between Ras2 and the Ira proteins. We also provide evidence that the two Ras-binding sites mapped on Cyr1p are required for the signalling complex assembly. In fact, we show that the cyr1Δ strain expressing CYR1 alleles lacking either the LRR region or the C-terminal domain still have a high basal and glucose-induced Ras2-GTP level. In contrast, a mutant expressing a Cyr1 protein only missing the N-terminal domain showed a normal Ras2 activation pattern. Likewise, the Ras2-GTP levels are comparable in the wild type strain and the srv2Δ strain, supporting the hypothesis that Cap is not essential for the Ras-adenylate cyclase interaction.

  2. Characterization of the purine-reactive site of the rat testis cytosolic adenylate cyclase.

    PubMed

    Onoda, J M; Braun, T; Wrenn, S M

    1987-06-15

    Naturally soluble rat germ cell adenylate cyclase was inhibited by adenosine and the adenosine analogs, 9-beta-D-arabinofuranosyl adenine (AFA) and 2',5'-dideoxyadenosine (DDA), all of which inhibited hormone-sensitive adenylate cyclases at the "P" site. The IC50 values for adenosine and DDA were approximately 0.1 and for AFA, 4.0 mM. The onset of adenosine inhibition was very rapid whether adenosine was added to the enzyme reactant mixture at time zero concomitantly with the addition of substrate or after the enzyme had been activated by the addition of substrate. The adenosine analogs, N6-methyladenosine (MeA) and N6-phenylisopropyl adenosine (PIA), which interact with plasma membrane receptors ("R" receptors) for hormone-sensitive adenylate cyclase, had little effect on the activity of the cytosolic adenylate cyclase. Additionally, aminophylline, which has been shown to competitively antagonize adenosine interactions with the plasma membrane "R" receptors but not "P" site interactions, had no effect upon substrate activation of the soluble enzyme and did not prevent adenosine from inhibiting the activity of the enzyme. These data provide evidence for an adenosine regulatory site on the cytosolic enzyme which resembles the "P" site described for membrane bound-adenylate cyclase.

  3. Adenylate cyclase regulation in the spermatogenic cell plasma membrane: Modulating effects of TPA and TCDD

    SciTech Connect

    Beebe, L.E.

    1989-01-01

    This research was designed to compare the effects of TPA, a phorbol ester, and TCDD in a spermatogenic cell population, a target of TCDD toxicity. Membrane-bound adenylate cyclase activity was used an index of membrane function, and was quantified by the amount of {sup 32}P-cAMP formed from {sup 32}P-ATP following chromatographic separation. Exposure to male germ cells in-vitro to TPA and TCDD followed by direct measurement of enzyme activity was used to investigate the potential of each agent to perturb membrane function. TPA and TCDD consistently inhibited adenylate cyclase activity at the levels of G{sub s}-catalytic unit coupling and hormone-receptor activation, as measured by the stimulation of enzyme activity by concomitant addition of forskolin and GTP and FSH and GTP, respectively. The effect on coupling required at least 60 minutes of exposure to TPA or TCDD. Concentration-response curves demonstrated a progressive desensitization with increasing TPA concentration, while TCDD exhibited consistent inhibition over the same concentration range.

  4. Bordetella adenylate cyclase toxin: a unique combination of a pore-forming moiety with a cell-invading adenylate cyclase enzyme.

    PubMed

    Masin, Jiri; Osicka, Radim; Bumba, Ladislav; Sebo, Peter

    2015-11-01

    The adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) is a key virulence factor of the whooping cough agent Bordetella pertussis. CyaA targets myeloid phagocytes expressing the complement receptor 3 (CR3, known as αMβ2 integrin CD11b/CD18 or Mac-1) and translocates by a poorly understood mechanism directly across the cytoplasmic membrane into cell cytosol of phagocytes an adenylyl cyclase(AC) enzyme. This binds intracellular calmodulin and catalyzes unregulated conversion of cytosolic ATP into cAMP. Among other effects, this yields activation of the tyrosine phosphatase SHP-1, BimEL accumulation and phagocyte apoptosis induction. In parallel, CyaA acts as a cytolysin that forms cation-selective pores in target membranes. Direct penetration of CyaA into the cytosol of professional antigen-presenting cells allows the use of an enzymatically inactive CyaA toxoid as a tool for delivery of passenger antigens into the cytosolic pathway of processing and MHC class I-restricted presentation, which can be exploited for induction of antigen-specific CD8(+) cytotoxic T-lymphocyte immune responses.

  5. Localization of nigrostriatal dopamine receptor subtypes and adenylate cyclase

    SciTech Connect

    Filloux, F.; Dawson, T.M.; Wamsley, J.K.

    1988-04-01

    Quantitative autoradiography using (/sup 3/H)-SCH 23390, (/sup 3/H)-sulpiride and (/sup 3/H)-forskolin was used to assess the effects of single and combined neurotoxin lesions of the nigrostriatal pathway in the rat brain on dopamine (DA) receptor subtypes and adenylate cyclase (AC), respectively. Ibotenic acid (IA) lesions of the caudate-putamen (CPu) resulted in near total loss of both (/sup 3/H)-SCH 23390 and of (/sup 3/H)-forskolin binding in the ipsilateral CPu and substantia nigra reticulata (SNR). (/sup 3/H)-sulpiride binding in the CPu was only partially removed by this same lesion, and nigral (/sup 3/H)-sulpiride binding was virtually unchanged. 6-Hydroxydopamine (6-OHDA) and IA lesions of the substantia nigra compacta (SNC) did not affect (/sup 3/H)-SCH 23390 or (/sup 3/H)-forskolin binding, but largely removed (/sup 3/H)-sulpiride binding in the SNC. A 6-OHDA lesion of the nigrostriatal pathway followed by an ipsilateral IA injection of the CPu failed to further reduce (/sup 3/H)-sulpiride binding in the CPu. These results demonstrate that postsynaptic DA receptors in the CPu are of both the D1 and D2 variety; however, a portion of D2 receptors in the CPu may be presynaptic on afferent nerve terminals to this structure. D1 receptors in the SNR are presynaptic on striatonigral terminals, whereas the D2 receptors of the SNC are autoreceptors on nigral DA neurons. The existence of presynaptic D2 receptors on nigrostriatal DA-ergic terminals could not be confirmed by this study. Co-localization of D1 receptors and AC occurs in both the CPu and SNR.

  6. Adenylate cyclase regulates elongation of mammalian primary cilia

    SciTech Connect

    Ou, Young; Ruan, Yibing; Cheng, Min; Moser, Joanna J.; Rattner, Jerome B.; Hoorn, Frans A. van der

    2009-10-01

    The primary cilium is a non-motile microtubule-based structure that shares many similarities with the structures of flagella and motile cilia. It is well known that the length of flagella is under stringent control, but it is not known whether this is true for primary cilia. In this study, we found that the length of primary cilia in fibroblast-like synoviocytes, either in log phase culture or in quiescent state, was confined within a range. However, when lithium was added to the culture to a final concentration of 100 mM, primary cilia of synoviocytes grew beyond this range, elongating to a length that was on average approximately 3 times the length of untreated cilia. Lithium is a drug approved for treating bipolar disorder. We dissected the molecular targets of this drug, and observed that inhibition of adenylate cyclase III (ACIII) by specific inhibitors mimicked the effects of lithium on primary cilium elongation. Inhibition of GSK-3{beta} by four different inhibitors did not induce primary cilia elongation. ACIII was found in primary cilia of a variety of cell types, and lithium treatment of these cell types led to their cilium elongation. Further, we demonstrate that different cell types displayed distinct sensitivities to the lithium treatment. However, in all cases examined primary cilia elongated as a result of lithium treatment. In particular, two neuronal cell types, rat PC-12 adrenal medulla cells and human astrocytes, developed long primary cilia when lithium was used at or close to the therapeutic relevant concentration (1-2 mM). These results suggest that the length of primary cilia is controlled, at least in part, by the ACIII-cAMP signaling pathway.

  7. Distribution of adenylate cyclase and GTP-binding proteins in hepatic plasma membranes.

    PubMed

    Dixon, B S; Sutherland, E; Alexander, A; Nibel, D; Simon, F R

    1993-10-01

    Hepatic membrane subfractions prepared from control rats demonstrated forskolin (FSK)-stimulated adenylate cyclase activity in the basolateral (sinusoidal) but not apical (canalicular) plasma membrane. After bile duct ligation (BDL) for 12 or 24 h, there was an increase in FSK-stimulated adenylate cyclase activity in the apical membrane (54.2 +/- 3.9 pmol.mg-1 x min-1). The mechanism for this increase was explored further. ATP hydrolysis was found to be much higher in the apical than the basolateral membrane. Increasing the ATP levels in the assay enhanced apical membrane adenylate cyclase activity (10.5 +/- 0.2 pmol.mg-l.min-1); however, total adenosinetriphosphatase (ATPase) activity was not altered after BDL. Extraction of the apical membrane with bile acids or other detergents resulted in a two- to threefold increase in adenylate cyclase activity (30.6 +/- 3.6 pmol.mg-1 x min-1; detergent C12E8) This suggested that bile duct ligation was acting via the detergent-like action of bile acids to uncover latent adenylate cyclase activity on apical membranes. Further studies demonstrated that both BDL and detergent extraction also enhanced toxin-directed ADP-ribosylation of Gs alpha (cholera toxin) and Gi alpha (pertussis toxin) in the apical but not the basolateral membrane. After BDL, Gi alpha was found to be twofold greater in the apical membrane than the basolateral membrane. Immunoblotting using specific G protein antibodies further confirmed that apical membranes from control rats had a higher concentration of Gi1, 2 alpha and beta and slightly elevated levels of Gi3 alpha and Gs alpha compared with the basolateral membrane. The results demonstrate that adenylate cyclase and heterotrimeric GTP-binding proteins are present on the apical membrane, but measurement of their functional activity requires detergent permeabilization of apical membrane vesicles and is limited by the presence of high ATPase activity.

  8. Dopamine inhibition of anterior pituitary adenylate cyclase is mediated through the high-affinity state of the D/sub 2/ receptor

    SciTech Connect

    Borgundvaag, B.; George, S.R.

    1985-07-29

    The diterpinoid forskolin stimulated adenylate cyclase activity (measured by conversion of (/sup 3/H)-ATP to (/sup 3/H)-cAMP) in anterior pituitary from male and female rats. Inhibition of stimulated adenylate cyclase activity by potent dopaminergic agonists was demonstrable only in female anterior pituitary. The inhibition of adenylate cyclase activity displayed a typically dopaminergic rank order of agonist potencies and could be completely reversed by a specific dopamine receptor antagonist. The IC/sub 50/ values of dopamine agonist inhibition of adenylate cyclase activity correlated with equal molarity with the dissociation constant of the high-affinity dopamine agonist-detected receptor binding site and with the IC/sub 50/ values for inhibition of prolactin secretion. These findings support the hypothesis that it is the high-affinity form of the D/sub 2/ dopamine receptor in anterior pituitary which is responsible for mediating the dopaminergic function of attenuating adenylate cyclase activity. 12 references, 4 figures, 1 table.

  9. [The aspects of adenylate cyclase activity regulation in myocardium cell membranes during hypokinesia].

    PubMed

    Bulanova, K Ia; Komar, E S; Lobanok, L M

    1999-01-01

    Nonstimulated and isoproterenol, GTF, GITF, NaF stimulated activities of the adenylate cyclase in sarcolemma in white rats' myocardium was studied after two weeks of hypokinesia. As was established, in restrained animals the sensitivity of adenylate cyclase to the specified agents was increased and transition to the bimodal GTF regulation took place. It is hypothesised that involvement of membrane-bound Gi-proteins in the adrenergic effects on cardiomyocytes is one of mechanisms of the cardiotropic effects of restraint and heart distresses.

  10. [Characteristics of interaction of adenylate cyclase modulators and phosphoinositide cell signaling systems with lipid langmuir monolayers].

    PubMed

    Liakhov, O M; Prokopenko, V V; Prokopenko, R A; Mohylevych, S Ie

    2006-01-01

    Interaction of two groups of bioregulators, which oppositely affect activity of adenylate cyclase and phosphoinositide cellular signaling systems, with the Langmuir monolayer films made of natural lecithin was studied. Most significant influence on the structural and energy characteristics of lipid monolayers was revealed for the group of bioregulators, which inhibit polyphosphoinositide signaling system or/and activate adenylate cyclase signaling system. It is shown, that using the cluster analysis the bioregulators can be divided into two groups according to general orientation of their action on the considered systems of transduction of a signal.

  11. 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.

  12. 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.; Seidler, F.J. )

    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. 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.

  13. The invasive adenylate cyclase of Bordetella pertussis. Properties and penetration kinetics.

    PubMed Central

    Friedman, E; Farfel, Z; Hanski, E

    1987-01-01

    Bordetella pertussis, the causative organism of whooping cough, produces a calmodulin-sensitive adenylate cyclase. Confer & Eaton [(1982) Science 217, 948-950] have shown that an extract from B. pertussis increases intracellular cyclic AMP levels in neutrophils and suggested that this increase is caused by the bacterial adenylate cyclase which penetrates these cells. We demonstrate in the present study that adenylate cyclase activity in lysates from lymphocytes exposed to a partially purified preparation of the bacterial enzyme has properties completely different from those of the intrinsic membrane-bound enzyme. Adenylate cyclase activity in lysates from lymphocytes exposed to the invasive enzyme is insensitive to N-ethylmaleimide, readily inactivated by acetic anhydride and relatively stable to SDS. Similar properties are exhibited by the bacterial enzyme itself. By contrast, the intrinsic membrane-bound enzyme activated by forskolin and guanosine 5'-gamma-thiotriphosphate is sensitive to N-ethylmaleimide and SDS and relatively stable to acetic anhydride. This strongly supports the notion that B. pertussis adenylate cyclase penetrates cells. Using the partially purified preparation of the invasive enzyme, we have studied the kinetics of its penetration. The intracellular catalytic activity reaches a steady state within 20 min, irrespective of enzyme or cell concentration. Steady-state levels are maintained for at least 2 h provided that the invasive enzyme is present in the incubation medium. Upon its removal, a rapid decrease (t1/2 approximately equal to 15 min) in the intracellular cyclase level is observed. This decrease reflects intracellular inactivation of the bacterial enzyme and is not caused by the release of the enzyme to the cell medium. PMID:2886119

  14. 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, which 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.

  15. Histamine-, norepinephrine-, and dopamine-sensitive central adenylate cyclases: effects of chlorpromazine derivatives and butaclamol.

    PubMed

    Palmer, G C; Wagner, H R; Palmer, S J; Manian, A A

    1978-06-01

    A series of recently available derivatives (quaternary and hydroxylated) of chlorpromazine (CPZ) and butaclamol were evaluated with respect to antagonism of norepinephrine- (NE) (rat cerebral cortex), dopamine- (DA) (rat striatum) and histamine- (H) sensitive (rabbit cerebral cortex) adenylate cyclases. With incubated tissue slices (rat and rabbit cortices) CPZ-CH3, 7-OH-CPZ-CH3, beta-OH-CPZ and butaclamol displayed a capacity to inhibit either NE- or H- induced accumulation of adenosine cyclic 3',5'-monophosphate (cAMP). With the broken cellular enzyme responsive to DA, rather potent inhibition of enzyme activity (IC50 less than 24 micron) occurred with butaclamol, beta-OH-CPZ, 7,8,beta-triOH-CPZ, 7,8-dioxo-beta-OH-CPZ and 3,7,8-triOH-CPZ. It is concluded that the metabolites of CPZ contribute to the central therapeutic and/or side effects of the parent compound.

  16. Hyaluronic acid as capacitation inductor: metabolic changes and membrane-associated adenylate cyclase regulation.

    PubMed

    Fernández, S; Córdoba, M

    2014-12-01

    The aim of this research was to study the effect of hyaluronic acid on bovine cryopreserved spermatozoa compared with heparin as regards the variation of capacitation induction, cellular oxidative metabolism and intracellular signal induced by membrane-associated adenylate cyclase to propose hyaluronic acid as a capacitation inductor. Heparin or hyaluronic acid and lysophosphatidylcholine were used to induce sperm capacitation and acrosome reaction, respectively. 2',5'-dideoxyadenosine was used as a membrane-associated adenylate cyclase inhibitor. The highest percentages of capacitated spermatozoa and live spermatozoa with acrosome integrity were obtained by incubating sperm for 60 min using 1000 μg/ml hyaluronic acid. In these conditions, capacitation induced by hyaluronic acid was lower compared with heparin; nonetheless both glycosaminoglycans promote intracellular changes that allow true acrosome reaction in vitro induced by lysophosphatidylcholine in bovine spermatozoa. Oxygen consumption in heparin-capacitated spermatozoa was significantly higher than in hyaluronic acid-treated spermatozoa. With all treatments, mitochondrial coupling was observed when a specific uncoupler of the respiratory chain was added. The inhibition of membrane-associated adenylate cyclase significantly blocked capacitation induction produced by hyaluronic acid, maintaining a basal sperm oxygen uptake in contrast to heparin effect in which both sperm parameters were inhibited, suggesting that the membrane-associated adenylate cyclase activation is involved in the intracellular signal mechanisms induced by both capacitation inductors, but only regulates mitochondrial oxidative phosphorylation in heparin-capacitated spermatozoa.

  17. Functional consequences of single amino acid substitutions in calmodulin-activated adenylate cyclase of Bordetella pertussis.

    PubMed Central

    Glaser, P; Munier, H; Gilles, A M; Krin, E; Porumb, T; Bârzu, O; Sarfati, R; Pellecuer, C; Danchin, A

    1991-01-01

    Calmodulin-activated adenylate cyclase of Bordetella pertussis and Bacillus anthracis are two cognate bacterial toxins. Three short regions of 13-24 amino acid residues in these proteins exhibit between 66 and 80% identity. Site-directed mutagenesis of four residues in B. pertussis adenylate cyclase situated in the second (Asp188, Asp190) and third (His298, Glu301) segments of identity were accompanied by important decrease, or total loss, of enzyme activity. The calmodulin-binding properties of mutated proteins showed no important differences when compared to the wild-type enzyme. Apart from the loss of enzymatic activity, the most important change accompanying replacement of Asp188 by other amino acids was a dramatic decrease in binding of 3'-anthraniloyl-2'-deoxyadenosine 5'-triphosphate, a fluorescent analogue of ATP. From these results we concluded that the two neighbouring aspartic acid residues in B. pertussis adenylate cyclase, conserved in many other ATP-utilizing enzymes, are essential for binding the Mg(2+)-nucleotide complex, and for subsequent catalysis. Replacement of His298 and Glu301 by other amino acid residues affected the nucleotide-binding properties of adenylate cyclase to a lesser degree suggesting that they might be important in the mechanism of enzyme activation by calmodulin, rather than being involved directly in catalysis. PMID:2050107

  18. Topographic separation of adenylate cyclase and hormone receptors in the plasma membrane of toad erythrocyte ghosts

    PubMed Central

    Sahyoun, N.; Hollenberg, M. D.; Bennett, V.; Cuatrecasas, P.

    1977-01-01

    Brief sonication of whole erythrocyte plasma membranes (ghosts) from toads at 4° does not inactivate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1] or destroy the receptor binding properties of hydroxybenzylpindolol or insulin. The hormonal (but not the fluoride-induced) stimulation of this enzyme is, however, lost. Fractionation of the small, resealed membrane fragments (vesicles) on discontinuous sucrose gradients results in the separation of vesicle populations differing grossly in size and protein composition. In addition, the distribution of the β-adrenergic receptor, an insulin binding site, and adenylate cyclase among these vesicles fractions differs. The pattern of distribution of these functional structures can be altered differentially by manipulations of the ghosts before sonication. For example, brief preincubation with isoproterenol leads to a change in the relative distribution of β-receptor (but not adenylate cyclase) among the various vesicle fractions; this effect is not obtained with β-receptor antagonists, which block the isoproterenol effect. Exposure of the ghosts to different temperatures, changes in the divalent cation composition of the medium, or the addition of ATP also leads to changes in the distribution of surface markers of the subsequently formed vesicles. The results indicate gross asymmetries in the distribution of protein components within the plane of the membrane and raise important questions regarding the manner whereby functionally related and coupled components, such as hormone receptors and adenylate cyclase, interact. Images PMID:197522

  19. Modulation of receptors and adenylate cyclase activity during sucrose feeding, food deprivation, and cold exposure

    SciTech Connect

    Scarpace, P.J.; Baresi, L.A.; Morley, J.E. Univ. of California, Los Angeles )

    1987-12-01

    Thermogenesis in brown adipose tissue (BAT) serves as a regulator of body temperature and weight maintenance. Thermogenesis can be stimulated by catecholamine activation of adenylate cyclase through the {beta}-adrenergic receptor. To investigate the effects of sucrose feeding, food deprivation, and cold exposure on the {beta}-adrenergic pathway, adenylate cyclase activity and {beta}-adrenergic receptors were assessed in rat BAT after 2 wk of sucrose feeding, 2 days of food deprivation, or 2 days of cold exposure. {beta}-Adrenergic receptors were identified in BAT using ({sup 125}I)iodocyanopindolol. Binding sites had the characteristics of mixed {beta}{sub 1}- and {beta}{sub 2}-type adrenergic receptors at a ratio of 60/40. After sucrose feeding or cold exposure, there was the expected increase in BAT mitochondrial mass as measured by total cytochrome-c oxidase activity but a decrease in {beta}-adrenergic receptor density due to a loss of the {beta}{sub 1}-adrenergic subtype. This BAT {beta}-adrenergic receptor downregulation was tissue specific, since myocardial {beta}-adrenergic receptors were unchanged with either sucrose feeding or cold exposure. Forskolin-stimulated adenylate cyclase activity increased in BAT after sucrose feeding or cold exposure but not after food deprivation. These data suggest that in BAT, sucrose feeding or cold exposure result in downregulation of {beta}-adrenergic receptors and that isoproterenol-stimulated adenylate cyclase activity was limited by receptor availability.

  20. Inhibitors of receptor-mediated endocytosis block the entry of Bacillus anthracis adenylate cyclase toxin but not that of Bordetella pertussis adenylate cyclase toxin.

    PubMed Central

    Gordon, V M; Leppla, S H; Hewlett, E L

    1988-01-01

    Bordetella pertussis and Bacillus anthracis produce extracytoplasmic adenylate cyclase toxins (AC toxins) with shared features including activation by calmodulin and the ability to enter target cells and catalyze intracellular cyclic AMP (cAMP) production from host ATP. The two AC toxins were evaluated for sensitivities to a series of inhibitors of known uptake mechanisms. Cytochalasin D, an inhibitor of microfilament function, abrogated the cAMP response to B. anthracis AC toxin (93%) but not the cAMP response elicited by B. pertussis AC toxin. B. anthracis-mediated intoxication of CHO cells was completely inhibited by ammonium chloride (30 mM) and chloroquine (0.1 mM), whereas the cAMP accumulation produced by B. pertussis AC toxin remained unchanged. The block of target cell intoxication by cytochalasin D could be bypassed when cells were first treated with anthrax AC toxin and then exposed to an acidic medium. These data indicate that despite enzymatic similarities, these two AC toxins intoxicate target cells by different mechanisms, with anthrax AC toxin entering by means of receptor-mediated endocytosis into acidic compartments and B. pertussis AC toxin using a separate, and as yet undefined, mechanism. PMID:2895741

  1. Iodide-induced inhibition of adenylate cyclase activity in horse and dog thyroid.

    PubMed

    Cochaux, P; Van Sande, J; Swillens, S; Dumont, J E

    1987-12-30

    The characteristics of the iodide-induced inhibition of cyclic AMP accumulation in dog thyroid slices have been previously described [Van Sande, J., Cochaux, P. and Dumont, J. E. (1985) Mol. Cell. Endocrinol. 40, 181-192]. In the present study we investigated the characteristics of the iodide-induced inhibition of adenylate cyclase activity in dog and horse thyroid. The inhibition of cyclic AMP accumulation by iodide in stimulated horse thyroid slices was similar to that observed in dog thyroid slices. The inhibition was observed in slices stimulated by thyroid-stimulating hormone, cholera toxin and forskolin. Increasing the concentration of the stimulators did not overcome the iodide-induced inhibition. Adenylate cyclase activity, assayed in crude homogenates or in plasma-membrane-containing particulates (100,000 x g pellets), was lower in homogenates or in particulates prepared from iodide-treated slices than from control slices. This inhibition was observed on the cyclase activity stimulated by forskolin, fluoride or guanosine 5'-[beta, gamma-imino]triphosphate, but also on the basal activity. It was relieved when the homogenate was prepared from slices incubated with iodide and methimazole. Similar results were obtained with dog thyroid. The inhibition persisted when the particulate fraction was washed three times during 1 h at 100,000 x g, in the presence of bovine serum albumin or increasing concentration of KCl. It was similar whatever the duration of the cyclase assay, in a large range of protein concentration. These results indicate that a stable modification of adenylate cyclase activity, closely related to the plasma membrane, was induced when slices were incubated with iodide. Iodide inhibition did not modify the affinity of adenylate cyclase for its substrate (MgATP), but induced a decrease of the maximal velocity of the enzyme. The percentage inhibition was slightly decreased when Mg2+ concentration increased, and markedly decreased when Mn2

  2. Alkaline phosphatase relieves desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocyte membranes

    SciTech Connect

    Stadel, J.M.; Rebar, R.; Crooke, S.T.

    1987-05-01

    Desensitization of adenylate cyclase-coupled ..beta..-adrenergic receptors in avian erythrocytes results in 40-65% decrease in agonist-stimulated adenylate cyclase activity and correlates with increased phosphorylation of ..beta..-adrenergic receptors. To assess the role of phosphorylation in desensitization, membranes from isoproterenol- and cAMP-desensitized turkey erythrocytes were incubated with alkaline phosphatase for 30 min at 37/sup 0/C, pH = 8.0. In both cases alkaline phosphatase treatment significantly reduced desensitization of agonist-stimulated adenylate cyclase activity by 40-60%. Similar results were obtained following alkaline phosphatase treatment of membranes from isoproterenol- and cAMP-desensitized duck erythrocytes. In addition, alkaline phosphatase treatment of membranes from duck erythrocytes desensitized with phorbol 12-mystrate 13-acetate returned adenylate cyclase activity to near control values. In all experiments inclusion of 20 mM NaPO/sub 4/ to inhibit alkaline phosphatase during treatment of membranes blocked the enzyme's effect on agonist-stimulated adenylate cyclase activity. These results demonstrate a role for phosphorylation in desensitization of adenylate cyclase-coupled ..beta..-adrenergic receptors in avian erythrocytes.

  3. Influence of volatile anesthetics on muscarinic receptor adenylate cyclase coupling in brain and heart

    SciTech Connect

    Anthony, B.L.

    1988-01-01

    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 affinity ({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.

  4. Adenylate cyclase, cyclic AMP and extracellular-signal-regulated kinase-2 in airway smooth muscle: modulation by protein kinase C and growth serum.

    PubMed Central

    Moughal, N; Stevens, P A; Kong, D; Pyne, S; Pyne, N J

    1995-01-01

    Bradykinin and phorbol 12-myristate 13-acetate stimulate adenylate cyclase activity in serum-depleted cultured airway smooth muscle via a protein kinase C (PKC)-dependent pathway. The probable target is the type II adenylate cyclase, which can integrate coincident signals from both PKC and Gs. Therefore, activation of Gs (by cholera-toxin pre-treatment) amplified the bradykinin-stimulated cyclic AMP signal and concurrently attenuated the partial activation of extracellular-signal-regulated kinase-2 (ERK-2) by bradykinin. We have previously demonstrated that, in order to induce full activation of ERK-2 with bradykinin, it is necessary to obliterate PKC-stimulated cyclic AMP formation. We concluded that the cyclic AMP signal limits the magnitude of ERK-2 activation [Pyne, Moughal, Stevens, Tolan and Pyne (1994) Biochem. J. 304, 611-616]. The present study indicates that the bradykinin-stimulated ERK-2 pathway is entirely cyclic AMP-sensitive, and suggests that coincident signal detection by adenylate cyclase may be an important physiological route for the modulation of early mitogenic signalling. Furthermore, the direct inhibition of adenylate cyclase activity enables bradykinin to induce DNA synthesis, indicating that the PKC-dependent activation of adenylate cyclase limits entry of cells into the cell cycle. These studies suggest that the mitogenicity of an agonist may be governed, in part, by its ability to stimulate an inhibitory cyclic AMP signal pathway in the cell. The activation of adenylate cyclase by PKC appears to be downstream of phospholipase D. However, in cells that were maintained in growth serum (i.e. were not growth-arrested), bradykinin was unable to elicit a PKC-stimulated cyclic AMP response. The lesion in the signal-response coupling was not at the level of either the receptor or phospholipase D, which remain functionally operative and suggests modification occurs at either PKC or adenylate cyclase itself. These studies are discussed with

  5. Muscarinic receptor binding and muscarinic receptor-mediated inhibition of adenylate cyclase in rat brain myelin

    SciTech Connect

    Larocca, J.N.; Ledeen, R.W.; Dvorkin, B.; Makman, M.H.

    1987-12-01

    High-affinity muscarinic cholinergic receptors were detected in myelin purified from rat brain stem with use of the radioligands /sup 3/H-N-methylscopolamine (/sup 3/H-NMS), /sup 3/H-quinuclidinyl benzilate (/sup 3/H-QNB), and /sup 3/H-pirenzepine. /sup 3/H-NMS binding was also present in myelin isolated from corpus callosum. In contrast, several other receptor types, including alpha 1- and alpha 2-adrenergic receptors, present in the starting brain stem, were not detected in myelin. Based on Bmax values from Scatchard analyses, /sup 3/H-pirenzepine, a putative M1 selective ligand, bound to about 25% of the sites in myelin labeled by /sup 3/H-NMS, a nonselective ligand that binds to both M1 and M2 receptor subtypes. Agonist affinity for /sup 3/H-NMS binding sites in myelin was markedly decreased by Gpp(NH)p, indicating that a major portion of these receptors may be linked to a second messenger system via a guanine-nucleotide regulatory protein. Purified myelin also contained adenylate cyclase activity; this activity was stimulated several fold by forskolin and to small but significant extents by prostaglandin E1 and the beta-adrenergic agonist isoproterenol. Myelin adenylate cyclase activity was inhibited by carbachol and other muscarinic agonists; this inhibition was blocked by the antagonist atropine. Levels in myelin of muscarinic receptors were 20-25% and those of forskolin-stimulated adenylate cyclase 10% of the values for total particulate fraction of whole brain stem. These levels in myelin are appreciably greater than would be predicted on the basis of contamination. Also, additional receptors and adenylate cyclase, added by mixing nonmyelin tissue with whole brain stem, were quantitatively removed during the purification procedure.

  6. Properties of the separated catalytic and regulatory units of brain adenylate cyclase.

    PubMed Central

    Strittmatter, S; Neer, E J

    1980-01-01

    Adenylate cyclase from bovine brain cortex was solubilized with 14 mM cholate and 1 M (NH4)2SO4. Gel filtration over a column of Sepharose 6B separated the catalytic unit (CU) from a factor (G/F) that confers responsiveness to 5'-guanylyl imidophosphate (p[NH]ppG) or fluoride. The separated CU, which elutes with a Kav, of 0.48 +/- 0.01 (n=5), is not responsive to p[NH]ppG or fluoride and is relatively inactive when Mg . ATP is the substrate but activated 8-15-fold by Mn2+. The separated G/F elutes with a Kav of 0.70 +/- 0.02 (n=4). It restores the responsiveness of the CU to p[NH]ppG and fluoride. Activation of the enzyme by p[NH]ppG before solubilization does not decrease the amount of G/F eluting with a Kav of 0.7. Therefore, the G/F is probably present in brain cortex in excess over the CU. p[NH]ppG stabilizes the G/F but not the CU against thermal inactivation, suggesting that it interacts with G/F and not with CU. Incubation of the G/F with p[NH]ppG before addition of CU markedly increases the rate of activation of the reconstituted enzyme by p[NH]ppG. We propose, therefore, that the rate-limiting step in adenylate cyclase activation is a process in G/F alone and not a slow conformational change in CU or a slow association of G/F with CU. Binding of p[NH]ppG to the isolated G/F appears to be readily reversible; the ability of fully activated G/F to stimulate CU can be blocked if GDP is added before CU. In contrast, after the CU has been activated by interaction with G/F, GDP cannot reverse the activation. This suggests that association with the CU increases the affinity of G/F for p[NH]ppG. PMID:6935648

  7. Relationship between muscarinic receptor occupancy and adenylate cyclase inhibition in the rabbit myocardium

    SciTech Connect

    Ehlert, F.J.

    1985-11-01

    The muscarinic receptor-binding properties of a series of muscarinic drugs were compared with their effects on adenylate cyclase in membranes of the rabbit myocardium. When measured by competitive inhibition of (TH)-N-methylscopolamine binding, the competition curves of the various agonists were adequately described by the ternary complex model. This model assumes that the receptor can bind reversibly with a guanine nucleotide binding protein in the membrane and that the affinity of the agonist for the receptor-guanine nucleotide-binding protein complex is higher than that for the free receptor. A satisfactory fit of the ternary complex model to the data could only be achieved assuming that very little receptor is precoupled with the guanine nucleotide-binding protein in the absence of agonist. There was good agreement between the efficacy of each agonist as measured by inhibition of adenylate cyclase and the estimate of the positive cooperativity between the binding of the agonist receptor complex and the guanine nucleotide-binding protein. Guanosine 5'-triphosphate (0.1 mM) had no significant effect on the binding of (TH)N-methylscopolamine but caused an increase in the concentration of the various agonists required for half-maximal receptor occupancy. There was good correlation between efficacy as measured by inhibition of adenylate cyclase and the influence of guanosine 5'-triphosphate on binding properties.

  8. Persistent stimulation of adenylate cyclase and urea transport by an AVP photolabel

    SciTech Connect

    Eggena, P.; Ma, C.L.; Fahrenholz, F.; Schwartz, I.L.

    1985-07-01

    The effects of a photoaffinity label for arginine vasopressin receptors, (Phe2, Phe(p-N3)3)AVP (N3-AVP), on urea permeability and adenylate cyclase activity have been investigated in the toad urinary bladder. This compound, when activated by ultraviolet light, induced a maximal and persistent increase in the urea permeability of the intact bladder and a persistent increase in the adenylate cyclase activity of toad bladder epithelial cell homogenates. Covalent attachment of the analogue to target tissue during photolysis was equivalent at 4 and 20 degrees C. Bladders exposed to N3-AVP in the presence of AVP during photolysis were substantially less permeable to urea than controls that had been exposed to N3-AVP alone. These findings constitute further evidence in support of the previous suggestion that N3-AVP binds covalently to AVP receptors and, in addition, demonstrates that N3-AVP evokes a persistent increase in adenylate cyclase activity which, in turn, triggers a persistent increase in bladder permeability to urea.

  9. Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in Syrian hamster hypothalamus

    SciTech Connect

    Niles, L.P.; Hashemi, F. )

    1990-12-01

    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 = 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.

  10. 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 not 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.

  11. The invasive adenylate cyclase of Bordetella pertussis. Intracellular localization and kinetics of penetration into various cells.

    PubMed Central

    Farfel, Z; Friedman, E; Hanski, E

    1987-01-01

    The penetration of Bordetella pertussis adenylate cyclase into various mammalian cells exhibits similar kinetics; the accumulation of both intracellular cyclase activity and cyclic AMP is rapid, reaching constant levels after 15-60 min of incubation. The kinetics of enzyme penetration into turkey erythrocytes is different; cyclase activity and cyclic AMP accumulate linearly and do not reach constant levels even after 6 h of incubation. In the preceding paper [Friedman, Farfel & Hanski (1987) Biochem. J. 243, 145-151] we have suggested that the constant level of intracellular cyclase activity reflects a steady state formed by continuous penetration and intracellular inactivation of the enzyme. In contrast with other mammalian cells, no inactivation of cyclase is observed in turkey erythrocytes. These results further support the notion that there is continuous penetration and deactivation of the invasive enzyme in mammalian cells. A 5-6-fold increase in specific activity of the invasive cyclase is detected in a pellet fraction of human lymphocytes in which a similar increase in specific activity of the plasma-membrane marker 5'-nucleotidase is observed. A similar increase in the invasive-cyclase specific activity is detected in a membrane fraction of human erythrocytes. Cyclase activity in a membrane-enriched fraction of human lymphocytes reached a constant level after 20 min of cell exposure to the enzyme. Similar time courses were observed for accumulation of cyclase activity and cyclic AMP in whole lymphocytes [Friedman, Farfel & Hanski (1987) Biochem, J. 243, 145-151]. We suggest therefore that cyclic AMP generation by the invasive enzyme as well as the intracellular inactivation process occur while it is associated with a membrane fraction identical, or closely associated, with the plasma membrane. PMID:2886120

  12. Effect of mitomycin C on the activation of adenylate cyclase in rat ascites hepatoma AH130 cells.

    PubMed

    Miyamoto, K; Matsunaga, T; Sanae, F; Koshiura, R

    1986-09-01

    Isoproterenol (IPN)-stimulated activity of adenylate cyclase was enhanced in a dose-dependent manner by exposure of AH130 cells to mitomycin C (MMC). The enhancement was also observed in prostaglandin E1-, guanine nucleotide analog-, NaF-, cholera toxin- and forskolin-stimulated activities of the enzyme but not in manganese-stimulated activity. In addition, even when the cells pretreated with islet-activating protein were exposed to MMC, IPN-stimulated activity of adenylate cyclase was enhanced. Anaerobic exposure of AH130 cells to MMC somewhat inhibited IPN-stimulated activity of adenylate cyclase in contrast with aerobic exposure. Exposure of cells to adriamycin also caused enhancement of IPN-stimulated activity of adenylate cyclase but exposure to nitrogen mustard inhibited the enzyme stimulation by IPN. The enhancing effect of MMC was lost by the combined treatment with alpha-tocopherol. From these results, it was shown that MMC modulated the activity of adenylate cyclase, probably through alterations in membrane structure.

  13. Inhibition of adenylate cyclase by delta 9-tetrahydrocannabinol in mouse spleen cells: a potential mechanism for cannabinoid-mediated immunosuppression.

    PubMed

    Schatz, A R; Kessler, F K; Kaminski, N E

    1992-01-01

    The ability of delta 9-Tetrahydrocannabinol (delta 9-THC) to modulate adenylate cyclase activity in mouse spleen cells was investigated. These studies were prompted by the recent identification and cloning of a G-protein coupled cannabinoid receptor localized in certain regions of the brain and the potential for a common mechanism between cannabinoid-mediated CNS effects and immunosuppression. Temporal addition studies were initially performed to identify the period of time when spleen cells in culture were most susceptible to the inhibitory effects of delta 9-THC, as measured by the day 5 IgM antibody forming cell response. delta 9-THC was only inhibitory when added to spleen cell cultures during the first 2 hr following antigen sensitization. In light of this time course, adenylate cyclase activity was measured in spleen cells incubated in the presence of 22 microM delta 9-THC for 5 min and subsequently stimulated with forskolin. delta 9-THC treated spleen cells demonstrated a 33% inhibition and a 66% inhibition in intracellular cAMP after a 5 or 15 min stimulation with forskolin, respectively. These studies suggest that inhibition of immune function by delta 9-THC may be mediated through the inhibition of intracellular cAMP early after antigen stimulation.

  14. Leucine-rich repeats and carboxyl terminus are required for interaction of yeast adenylate cyclase with RAS proteins.

    PubMed Central

    Suzuki, N; Choe, H R; Nishida, Y; Yamawaki-Kataoka, Y; Ohnishi, S; Tamaoki, T; Kataoka, T

    1990-01-01

    A Saccharomyces cerevisiae gene encoding adenylate cyclase has been analyzed by deletion and insertion mutagenesis to localize regions required for activation by the Sa. cerevisiae RAS2 protein. The NH2-terminal 657 amino acids were found to be dispensable for the activation. However, almost all 2-amino acid insertions in the middle 600 residues comprising leucine-rich repeats and deletions in the COOH-terminal 66 residues completely abolished activation by the RAS2 protein, whereas insertion mutations in the other regions generally had no effect. Chimeric adenylate cyclases were constructed by swapping the upstream and downstream portions surrounding the catalytic domains between the Sa. cerevisiae and Schizosaccharomyces pombe adenylate cyclases and examined for activation by the RAS2 protein. We found that the fusion containing both the NH2-terminal 1600 residues and the COOH-terminal 66 residues of the Sa. cerevisiae cyclase rendered the catalytic domain of the Sc. pombe cyclase, which otherwise did not respond to RAS proteins, activatable by the RAS2 protein. Thus the leucine-rich repeats and the COOH terminus of the Sa. cerevisiae adenylate cyclase appear to be required for interaction with RAS proteins. Images PMID:2247439

  15. Cellular levels of feedback regulator of adenylate cyclase and the effect of epinephrine and insulin.

    PubMed Central

    Ho, R j; Russell, T R; Asakawa, T; Sutherland, E W

    1975-01-01

    We have obtained direct evidence that shows the cellular formation and subsequent release of a potent inhibitor (feedback regulator) of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by adipocytes, upon stimulation with epinephrine. The appearance of such a feedback regulator in adipocytes preceded its release into the medium. During a 30 min incubation, intracellular regulator levels rose rapidly and reached 39-61 units/g of adipocyte at 10 min. Release of inhibitor into the medium increased slowly and was 11-16 units/g of adipocyte at 10 min. Upon continued incubation, the cells at 30 min contained 30-41 units/g of ingibitor, slightly less than the content at 30 min; meanwhile, the medium content rose more than 3-fold. The inhibitor from both locations appeared to have the same characteristics, judging from the purification procedures and the biological activities on hormone-stimulated adenylate cyclase. Adenylate cyclase was inhibited by the feedback regulator in vitro when either epinephrine, corticotropin (ACTH), or glucagon was used as activator. The site of action of this inhibitor is therefore most likely beyond the specific hormone receptors. A new in vitro action of insulin has been found. Insulin, 50-500 microunits/ml, inhibited the formation and release of this factor from isolated rat or hamster adipocytes by 29-81% after these cells were stimulated by hormones that raise intracellular adenosine 3':5'-cyclic monophosphate. This factor enhaced the effect of insulin in lowering the adenosine 3':5'-cyclic monophosphate levels in fresh rat adipocytes. A reduced formation of such a factor may modify the metabolic events in adipocytes, and some as yet unexplained effects of insulin could therefore be linked to the metabolic effects of this factor. PMID:174073

  16. Pituitary adenylate cyclase-activating polypeptide is a sympathoadrenal neurotransmitter involved in catecholamine regulation and glucohomeostasis.

    PubMed

    Hamelink, Carol; Tjurmina, Olga; Damadzic, Ruslan; Young, W Scott; Weihe, Eberhard; Lee, Hyeon-Woo; Eiden, Lee E

    2002-01-08

    The adrenal gland is important for homeostatic responses to metabolic stress: hypoglycemia stimulates the splanchnic nerve, epinephrine is released from adrenomedullary chromaffin cells, and compensatory glucogenesis ensues. Acetylcholine is the primary neurotransmitter mediating catecholamine secretion from the adrenal medulla. Accumulating evidence suggests that a secretin-related neuropeptide also may function as a transmitter at the adrenomedullary synapse. Costaining with highly specific antibodies against the secretin-related neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) and the vesicular acetylcholine transporter (VAChT) revealed that PACAP is found in nerve terminals at all mouse adrenomedullary cholinergic synapses. Mice with a targeted deletion of the PACAP gene had otherwise normal cholinergic innervation and morphology of the adrenal medulla, normal adrenal catecholamine and blood glucose levels, and an intact initial catecholamine secretory response to insulin-induced hypoglycemia. However, insulin-induced hypoglycemia was more profound and longer-lasting in PACAP knock-outs, and was associated with a dose-related lethality absent in wild-type mice. Failure of PACAP-deficient mice to adequately counterregulate plasma glucose levels could be accounted for by impaired long-term secretion of epinephrine, secondary to a lack of induction of tyrosine hydroxylase, normally occurring after insulin hypoglycemia in wild-type mice, and a consequent depletion of adrenomedullary epinephrine stores. Thus, PACAP is needed to couple epinephrine biosynthesis to secretion during metabolic stress. PACAP appears to function as an "emergency response" cotransmitter in the sympathoadrenal axis, where the primary secretory response is controlled by a classical neurotransmitter but sustained under paraphysiological conditions by a neuropeptide.

  17. Presence of hormonally-sensitive adenylate cyclase receptors in capillary-enriched fractions from rat cerebral cortex.

    PubMed

    Baca, G M; Palmer, G C

    1978-01-01

    The 10 000 g particulate fraction from capillary-enriched fractions isolated from rat cerebral cortex was shown to possess an adenylate cyclase highly sensitive to activation by sodium fluoride, norepinephrine, epinephrine, isoproterenol and dopamine. To a lesser extent histamine and three dopamine agonists, namely M-7 (5,6-dihydroxy-2-dimethylamino tetralin), ET-495 (methane sulfonate of pyribedil), and S-584 (metabolite of pyribedil) stimulated the enzyme preparation. The action of norepinephrine was blocked by propanolol while phenotolamine and haloperidol were relatively ineffective except at highest concentrations. Phentolamine and propanolol at only highest concentrations (10(-4) M) antagonized the action of dopamine. Haloperidol was seen to be a potent inhibitor of either dopamine- or dopamine agonist-sensitive adenylate cyclase. No effects on the enzyme were observed with methoxamine, octopamine or serotonin. These preliminary data suggest the presence of a mixed population of receptors for adenylate cyclase in rat brain capillaries.

  18. Rapid kinetics of 2-adrenergic agonist binding and inhibition of adenylate cyclase

    SciTech Connect

    Thomsen, W.; Neubig, R.R.

    1987-05-01

    Activation of 2-adrenergic receptors in human platelets results in inhibition of adenylate cyclase (AC). To elucidate the relation between agonist binding and response, the authors have used a novel rapid-mix quench method to compare the kinetics of binding and response. At functionally effective concentrations, the time course of binding of the full 2-agonist, (TH)UK14,304 (UK), to purified platelet membranes was faster than could be measured manually. Using the rapid-mix quench method, agonist binding was quantitated for times for 0.3 to 60 seconds. UK binding exhibited biexponential kinetics. The rate constant of the fast binding component increases linearly with agonist concentration from 1 to 100 nM with a second order rate constant and 7 x 10WM s (at 25C). The slow rate constant was nearly independent of agonist concentration. The half times of the fast and slow components of binding for 100 nM UK are 1.5 seconds and approximately 2 minutes respectively. The rate and magnitude of the fast binding was unaffected by 10 M GTP whereas the magnitude of the slow phase was markedly reduced. Inhibition of forskolin stimulated AC by 100 M epinephrine occurs with a lag of 5-10 seconds in the presence of 10 M GTP. At lower GTP concentrations, this lag is prolonged. The observation that the fast component of agonist binding precedes inhibition even at agonist concentrations 20-fold lower than the EC40 for responses indicates that the rate limiting step in inhibition of AC is distal to the binding of agonist.

  19. Pituitary adenylate cyclase activating polypeptide: an important vascular regulator in human skin in vivo.

    PubMed

    Seeliger, Stephan; Buddenkotte, Jörg; Schmidt-Choudhury, Anjona; Rosignoli, Carine; Shpacovitch, Victoria; von Arnim, Ulrike; Metze, Dieter; Rukwied, Roman; Schmelz, Martin; Paus, Ralf; Voegel, Johannes J; Schmidt, Wolfgang E; Steinhoff, Martin

    2010-11-01

    Pituitary adenylate cyclase-activating peptide (PACAP) is an important neuropeptide and immunomodulator in various tissues. Although this peptide and its receptors (ie, VPAC1R, VPAC2R, and PAC1R) are expressed in human skin, their biological roles are unknown. Therefore, we tested whether PACAP regulates vascular responses in human skin in vivo. When injected intravenously, PACAP induced a significant, concentration-dependent vascular response (ie, flush, erythema, edema) and mediated a significant and concentration-dependent increase in intrarectal body temperature that peaked at 2.7°C. Topical application of PACAP induced marked concentration-dependent edema. Immunohistochemistry revealed a close association of PACAP-immunoreactive nerve fibers with mast cells and dermal blood vessels. VPAC1R was expressed by dermal endothelial cells, CD4+ and CD8+ T cells, mast cells, and keratinocytes, whereas VPAC2R was expressed only in keratinocytes. VPAC1R protein and mRNA were also detected in human dermal microvascular endothelial cells. The PACAP-induced change in cAMP production in these cells demonstrated VPAC1R to be functional. PACAP treatment of organ-cultured human skin strongly increased the number of CD31+ vessel cross-sections. Taken together, these results suggest that PACAP directly induces vascular responses that may be associated with neurogenic inflammation, indicating for the first time that PACAP may be a crucial vascular regulator in human skin in vivo. Antagonists to PACAP function may be beneficial for the treatment of inflammatory skin diseases with a neurogenic component.

  20. Desensitization of adenylate cyclase in a human keratinocyte cell line by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

    SciTech Connect

    Choi, E.J.; Young, M.J.; Toscano, D.L.; Greenlee, W.F.; Toscano, W.A. Jr.

    1987-05-01

    Regulation of adenylate cyclase in human keratinocyte cell line SCC 12 is altered after TCDD exposure. TCDD-treated cells show a 50% decrease in isoproterenol - stimulated adenylate cyclase activity. The reduced responsiveness of these cells to isoproterenol was concentration dependent on TCDD. The inactive TCDD analog, 2,7-dibenzo-p-dioxin did not affect isoproterenol activation. Altered hormone stimulation of adenylate cyclase can result from decreased receptor number or affinity, a defect in coupling of receptors via G/sub s/, or modification of the catalytic subunit. To distinguish between these possibilities, enzyme activity was assayed in the presence of different site-specific activators of this enzyme system. Cells exposed to TCDD for 24 hr showed a reduced response to the GTP analog, Gpp(NH)p. Forskolin stimulation was not affected by TCDD treatment. (/sup 125/I)-iodocyanopindolol (ICP) binding to ..beta..-adrenergic receptors was examined after TCDD treatment. The equilibrium dissociation constant (K/sub d/) for ICP was unaffected by TCDD treatment, whereas, the total number of specific ICP-binding sites was reduced from 1080 in control cells to 780 sites per cell in TCDD (10 nM) exposed cells.

  1. Adenylate Cyclase AcyA Regulates Development, Aflatoxin Biosynthesis and Fungal Virulence in Aspergillus flavus

    PubMed Central

    Yang, Kunlong; Qin, Qiuping; Liu, Yinghang; Zhang, Limei; Liang, Linlin; Lan, Huahui; Chen, Chihao; You, Yunchao; Zhang, Feng; Wang, Shihua

    2016-01-01

    Aspergillus flavus is one of the most important opportunistic pathogens of crops and animals. The carcinogenic mycotoxin, aflatoxins produced by this pathogen cause a health problem to human and animals. Since cyclic AMP signaling controls a range of physiological processes, like fungal development and infection when responding to extracellular stimuli in fungal pathogens, in this study, we investigated the function of adenylate cyclase, a core component of cAMP signaling, in aflatoxins biosynthesis and virulence on plant seeds in A. flavus. A gene replacement strategy was used to generate the deletion mutant of acyA that encodes the adenylate cyclase. Severe defects in fungal growth, sporulation and sclerotia formation were observed in the acyA deletion mutant. The defect in radical growth could be partially rescued by exogenous cAMP analog. The acyA mutant was also significantly reduced in aflatoxins production and virulence. Similar to the former studies in other fungi, The acyA mutant showed enhancing tolerance to oxidative stress, but more sensitive to heat stress. Overall, the pleiotropic defects of the acyA deletion mutant indicates that the cAMP-PKA pathway is involved in fungal development, aflatoxins biosynthesis and plant seed invasion in A. flavus. PMID:28066725

  2. Adenylate cyclase toxin (ACT) from Bordetella hinzii: characterization and differences from ACT of Bordetella pertussis.

    PubMed

    Donato, Gina M; Hsia, Hung-Lun J; Green, Candace S; Hewlett, Erik L

    2005-11-01

    Bordetella hinzii is a commensal respiratory microorganism in poultry but is increasingly being recognized as an opportunistic pathogen in immunocompromised humans. Although associated with a variety of disease states, practically nothing is known about the mechanisms employed by this bacterium. In this study, we show by DNA sequencing and reverse transcription-PCR that both commensal and clinical strains of B. hinzii possess and transcriptionally express cyaA, the gene encoding adenylate cyclase toxin (ACT) in other pathogenic Bordetella species. By Western blotting, we also found that B. hinzii produces full-length ACT protein in quantities that are comparable to those made by B. pertussis. In contrast to B. pertussis ACT, however, ACT from B. hinzii is less extractable from whole bacteria, nonhemolytic, has a 50-fold reduction in adenylate cyclase activity, and is unable to elevate cyclic AMP levels in host macrophages (nontoxic). The decrease in enzymatic activity is attributable, at least in part, to a decreased binding affinity of B. hinzii ACT for calmodulin, the eukaryotic activator of B. pertussis ACT. In addition, we demonstrate that the lack of intoxication by B. hinzii ACT may be due to the absence of expression of cyaC, the gene encoding the accessory protein required for the acylation of B. pertussis ACT. These results demonstrate the expression of ACT by B. hinzii and represent the first characterization of a potential virulence factor of this organism.

  3. 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 Bmax 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

  4. Increase in the amount of adenylate cyclase in rat gastrocnemius muscle after denervation

    SciTech Connect

    Hashimoto, K.; Watanabe, Y.; Uchida, S.; Yoshida, H.

    1989-01-01

    After section of the sciatic nerve, the basal adenylate cyclase (AC) activity in rat gastrocnemius muscle increased 6-7 times per membrane protein and about 2 times per whole muscle in the following 30 or 40 days. The AC activity in the muscle 30 days after denervation was increased about 4 times by folskolin. Calcitonin gene-related peptide (CGRP) also increased the adenylate cyclase activity in the denervated muscle. The binding of (/sup 3/H)-forskolin to cells isolated from gastrocnemius muscle was examined to determine the amount of AC molecules. Inhibition of (/sup 3/H)-forskolin binding by increasing amounts of unlabeled forskolin gave a sigmoid curve with a IC/sub 50/ value of 3/times/10/sup /minus/7/M. Results showed that the number of (/sup 3/H)-forskolin binding sites per cell was higher on the denervated side than on the control side, like the basal AC activity. The IC/sub 50/ values for inhibition by unlabeled forskolin of binding of (/sup 3/H)-forskolin were similar to muscles on the control and denervated sides. These results suggest that an increase in the AC activity induced by denervation was due to an increase in the numbers of AC molecules in the muscle.

  5. Pituitary Adenylate Cyclase-Activating Polypeptide Ameliorates Experimental Acute Ileitis and Extra-Intestinal Sequelae

    PubMed Central

    Schulze, Silvia; Fischer, André; Grundmann, Ursula; Alutis, Marie; Kühl, Anja A.; Tamas, Andrea; Toth, Gabor; Dunay, Miklos P.; Göbel, Ulf B.; Reglodi, Dora; Bereswill, Stefan

    2014-01-01

    Background The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) plays pivotal roles in immunity and inflammation. So far, potential immune-modulatory properties of PACAP have not been investigated in experimental ileitis. Methodology/Principal Findings Mice were perorally infected with Toxoplasma (T.) gondii to induce acute ileitis (day 0) and treated daily with synthetic PACAP38 from day 1 to 6 post infection (p.i.; prophylaxis) or from day 4 to 6 p.i. (therapy). Whereas placebo-treated control mice suffered from acute ileitis at day 7 p.i. and succumbed to infection, intestinal immunopathology was ameliorated following PACAP prophylaxis. PACAP-treated mice exhibited increased abundance of small intestinal FOXP3+ cells, but lower numbers of ileal T lymphocytes, neutrophils, monocytes and macrophages, which was accompanied by less ileal expression of pro-inflammatory cytokines such as IL-23p19, IL-22, IFN-γ, and MCP-1. Furthermore, PACAP-treated mice displayed higher anti-inflammatory IL-4 concentrations in mesenteric lymph nodes and liver and higher systemic anti-inflammatory IL-10 levels in spleen and serum as compared to control animals at day 7 p.i. Remarkably, PACAP-mediated anti-inflammatory effects could also be observed in extra-intestinal compartments as indicated by reduced pro-inflammatory mediator levels in spleen (TNF-α, nitric oxide) and liver (TNF-α, IFN-γ, MCP-1, IL-6) and less severe histopathological sequelae in lungs and kidneys following prophylactic PACAP treatment. Strikingly, PACAP prolonged survival of T. gondii infected mice in a time-of-treatment dependent manner. Conclusion/Significance Synthetic PACAP ameliorates acute small intestinal inflammation and extra-intestinal sequelae by down-regulating Th1-type immunopathology, reducing oxidative stress and up-regulating anti-inflammatory cytokine responses. These findings provide novel potential treatment options of inflammatory bowel diseases. PMID:25238233

  6. Biochemical mechanisms of myocardial adenylate cyclase subsensitivity to isoproterenol in cardiac hypertrophy of spontaneously hypertensive rats

    SciTech Connect

    Cheon, J.W.

    1986-01-01

    The responsiveness of the myocardial adenylate cyclase (AC) system in generating cAMP was studied using isoproterenol (a beta-adrenergic receptor agonist), cholera toxin (a guanosinetriphosphatase inhibitor) and forskolin (a catalytic unit activator) in isolated myocytes of age-matched, 14-17 weeks old Wistar Kyoto normotensive rates (WKYs) and spontaneously hypertensive rats (SHRs). We found a reduction in isoproterenol-stimulated cAMP formation in myocytes of SHRs compared with WKYs. This reduction was not due to changes in isoproterenol-receptor interactions. Scatchard plot analysis of (/sup 3/H)CGP 12177 binding to beta-adrenergic receptors in isolated myocytes of WKYs and SHRs revealed to significant differences in the maximum number of binding sites or dissociation constant. There were no significant differences in Ki and IC/sub 50/ calculated from the competitive displacement of (/sup 3/H)CGP 12177 binding by (-) isoproterenol, suggesting no change in the affinity of the beta-adrenergic receptors for isoproterenol. We found no significant differences in forskolin-stimulated cAMP formation between the two groups. This suggest that the reduction in isoproterenol-stimulated cAMP formation observed in myocytes of SHRs is not due to changes in the ability of catalytic unit to convert ATP to cAMP. Interestingly, cholera toxin-stimulated cAMP formation was increased in myocytes of SHRs. One possible explanation for these observations may be increased guanosinetriphosphatase (GTPase) activation by isoproterenol in myocytes of SHRs. The activation of GTPase by isoproterenol in myocytes of SHRs. The activation of GTPase by isoproterenol was measured as the release of Pi from (..gamma..-/sup 32/P)GTP. There was an increase in isoproterenol-stimulated GTPase activity in myocytes of SHRs compared with WKYs.

  7. Homologous desensitization of adenylate cyclase: the role of. beta. -adrenergic receptor phosphorylation and dephosphorylation

    SciTech Connect

    Sibley, D.R.; Strasser, R.H.; Daniel, K.; Lefkowitz, R.J.

    1986-03-05

    The authors utilized the frog erythrocyte (FE) as a ..beta..-adreneric receptor (..beta..AR) model system in which to study homologous desensitization. Preincubation with isoproterenol (ISO) leads to a 50% decline in ISO-stimulated adenylate cyclase (AC) activity without significant changes in basal, PGE/sub 1/-, NaF-, GppNHp-, forskolin-, or MnCl/sub 2/-stimulated AC activities. ISO treatment also induces the sequestration of ..beta..AR from the cell surface as evidenced by a 35% decline in (/sup 3/H)CGP-12177 binding sites on the surface of intact FE. Treatment of intact FE with ISO also promotes ..beta..AR phosphorylation to 2 mol PO/sub 4//mol of ..beta..AR. At 25/sup 0/C, the time courses of ISO-induced AC desensitization, ..beta..AR sequestration and ..beta..AR phosphorylation are identical occurring without a lag and exhibiting a t 1/2 of 30 min and a maximal response at 2.5 hrs. The sequestered ..beta..AR can be partially recovered upon cell lysis in a light membrane fraction (LMF), separable from the plasma membranes using sucrose gradients or differential centrifugation. ..beta..AR phosphorylation is reversed in the sequestered LMF exhibiting a PO/sub 4//..beta..AR stoichiometry of 0.7 mol/mol - similar to that observed under basal conditions. These data suggest that phosphorylation of ..beta..AR in the plasma membrane promotes their translocation away from the cell surface into a sequestered membrane domain where the phosphorylation is reversed, thus, enabling the return of ..beta..AR back to the cell surface and recoupling with AC.

  8. Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium

    PubMed Central

    Ohki, Mio; Sugiyama, Kanako; Kawai, Fumihiro; Tanaka, Hitomi; Nihei, Yuuki; Unzai, Satoru; Takebe, Masumi; Matsunaga, Shigeru; Adachi, Shin-ichi; Shibayama, Naoya; Zhou, Zhiwen; Koyama, Ryuta; Takahashi, Tetsuo; Tame, Jeremy R. H.; Iseki, Mineo; Park, Sam-Yong

    2016-01-01

    Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit. PMID:27247413

  9. Pituitary Adenylate Cyclase-Activating Polypeptide induces a depressive-like phenotype in rats

    PubMed Central

    Seiglie, Mariel P.; Smith, Karen L.; Blasio, Angelo; Cottone, Pietro; Sabino, Valentina

    2015-01-01

    Major Depressive Disorder (MDD) is a chronic, life-threatening psychiatric condition characterized by depressed mood, psychomotor alterations, and a markedly diminished interest or pleasure in most activities, known as anhedonia. Available pharmacotherapies have limited success and the need for new strategies is clear. Recent studies attribute a major role to the pituitary adenylate cyclase-activating polypeptide (PACAP) system in mediating the response to stress. PACAP knockout mice display profound alterations in depressive-like behaviors and genetic association studies have demonstrated that genetic variants of the PACAP gene are associated with MDD. However, the effects of PACAP on depressive-like behaviors in rodents have not yet been systematically examined. The present study investigated the effects of central administration of PACAP in rats on depressive-like behaviors, using well-established animal models that represent some of the endophenotypes of depression. We used intracranial self-stimulation (ICSS) to assess the brain reward function, saccharin preference test to assess anhedonia, social interaction to assess social withdrawal, and forced swim test (FST) to assess behavioral despair. PACAP raised the current threshold for ICSS, elevation blocked by the PACAP antagonist PACAP(6-38). PACAP reduced the preference for a sweet saccharin solution, and reduced the time the rats spent interacting with a novel animal. Interestingly, PACAP administration did not affect immobility in the FST. Our results demonstrate a role for the central PACAP/PAC1R system in the regulation of depressive-like behaviors, and suggest that hyperactivity of the PACAP/PAC1R system may contribute to the pathophysiology of depression, particularly the associated anhedonic symptomatology and social dysfunction. PMID:26264905

  10. Hemodynamic actions of systemically injected pituitary adenylate cyclase activating polypeptide-27 in the rat

    NASA Technical Reports Server (NTRS)

    Whalen, E. J.; Johnson, A. K.; Lewis, S. J.

    1999-01-01

    The aims of this study were (1) to characterize the hemodynamic mechanisms underlying the hypotensive effects of pituitary adenylate cyclase activating polypeptide-27 (PACAP-27 0.1-2.0 nmol/kg, i.v.) in pentobarbital-anesthetized rats, and (2) to determine the roles of the autonomic nervous system, adrenal catecholamines and endothelium-derived nitric oxide (NO) in the expression of PACAP-27-mediated effects on hemodynamic function. PACAP-27 produced dose-dependent decreases in mean arterial blood pressure and hindquarter and mesenteric vascular resistances in saline-treated rats. PACAP-27 also produced pronounced falls in mean arterial blood pressure in rats treated with the ganglion blocker, chlorisondamine (5 mg/kg, i.v.). The hypotensive and vasodilator actions of PACAP-27 were not attenuated by the beta-adrenoceptor antagonist, propranolol (1 mg/kg, i.v.), or the NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME 50 micromol/kg, i.v.). PACAP-27 produced dose-dependent increases in heart rate whereas the hypotensive response produced by the nitrovasodilator, sodium nitroprusside (10 microg/kg, i.v.), was associated with a minimal tachycardia. The PACAP-27-induced tachycardia was unaffected by chlorisondamine, but was virtually abolished by propranolol. These results suggest that the vasodilator effects of PACAP-27 are due to actions in the microcirculation rather than to the release of adrenal catecholamines and that this vasodilation may not involve the release of endothelium-derived NO. These results also suggest that PACAP-27 produces tachycardia by directly releasing norepinephrine from cardiac sympathetic nerve terminals rather than by direct or baroreceptor reflex-mediated increases in sympathetic nerve activity.

  11. 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

  12. Cannabinoid inhibition of adenylate cyclase: relative activity of constituents and metabolites of marihuana.

    PubMed

    Howlett, A C

    1987-05-01

    delta 9Tetrahydrocannabinol (THC) has been shown to inhibit the activity of adenylate cyclase in the N18TG2 clone of murine neuroblastoma cells. The concentration of delta 9THC exhibiting half-maximal inhibition was 500 nM. delta 8Tetrahydrocannabinol was less active, and cannabinol was only partially active. Cannabidiol, cannabigerol, cannabichromene, olivetol and compounds having a reduced length of the C3 alkyl side chain were inactive. The metabolites of delta 8THC and delta 9THC hydroxylated at the C11 position were more potent than the parent drugs. However, hydroxylation at the C8 position of the terpenoid ring resulted in loss of activity. Compounds hydroxylated along the C3 alkyl side chain were equally efficacious but less potent than delta 9THC. These findings are compared to the pharmacology of cannabinoids reported for psychological effects in humans and behavioral effects in a variety of animal models.

  13. Crystal Structure of Human Soluble Adenylate Cyclase Reveals a Distinct, Highly Flexible Allosteric Bicarbonate Binding Pocket

    PubMed Central

    Saalau-Bethell, Susanne M; Berdini, Valerio; Cleasby, Anne; Congreve, Miles; Coyle, Joseph E; Lock, Victoria; Murray, Christopher W; O'Brien, M Alistair; Rich, Sharna J; Sambrook, Tracey; Vinkovic, Mladen; Yon, Jeff R; Jhoti, Harren

    2014-01-01

    Soluble adenylate cyclases catalyse the synthesis of the second messenger cAMP through the cyclisation of ATP and are the only known enzymes to be directly activated by bicarbonate. Here, we report the first crystal structure of the human enzyme that reveals a pseudosymmetrical arrangement of two catalytic domains to produce a single competent active site and a novel discrete bicarbonate binding pocket. Crystal structures of the apo protein, the protein in complex with α,β-methylene adenosine 5′-triphosphate (AMPCPP) and calcium, with the allosteric activator bicarbonate, and also with a number of inhibitors identified using fragment screening, all show a flexible active site that undergoes significant conformational changes on binding of ligands. The resulting nanomolar-potent inhibitors that were developed bind at both the substrate binding pocket and the allosteric site, and can be used as chemical probes to further elucidate the function of this protein. PMID:24616449

  14. Adrenalectomy mediated alterations in adrenergic activation of adenylate cyclase in rat liver

    SciTech Connect

    El-Refai, M.; Chan, T.

    1986-05-01

    Adrenalectomy caused a large increase in the number of ..beta..-adrenergic binding sites on liver plasma membranes as measured by /sup 125/I-iodocyanopindolol (22 and 102 fmol/mg protein for control and adrenalectomized (ADX) rats). Concomitantly an increase in the number of binding sites for /sup 3/H-yohimbine was also observed (104 and 175 fmol/mg protein for control and adx membranes). Epinephrine-stimulated increase in cyclic AMP accumulation in isolated hepatocytes were greater in cells from ADX rats. This increase in ..beta..-adrenergic mediated action was much less than what may be expected as a result of the increase in the ..beta..-adrenergic binding in ADX membranes. In addition phenoxybenzamine (10 ..mu..M) further augmented this action of epinephrine in both control and ADX cells. To test the hypothesis that the increase in the number of the inhibitory ..cap alpha../sub 2/-adrenergic receptors in adrenalectomy is responsible for the muted ..beta..-adrenergic response, the authors injected rats with pertussis toxin (PT). This treatment may cause the in vivo ribosylation of the inhibitory binding protein (Ni). Adenylate cyclase (AC) activity in liver plasma membranes prepared from treated and untreated animals was measured. In contrast with control rats, treatment of ADX rats with PT resulted in a significant increase in the basal activity of AC (5.5 and 7.7 pmol/mg protein/min for untreated and treated rats respectively). Isoproterenol (10 ..mu..M), caused AC activity to increase to 6.5 and 8.4 pmol/mg protein/min for membranes obtained from ADX untreated and ADX treated rats respectively. The ..cap alpha..-adrenergic antagonists had no significant effect on the ..beta..-adrenergic-mediated activation of AC in liver plasma membranes from PT treated control and ADX rats. The authors conclude that the ..beta..-adrenergic activation of AC is attenuated by Ni protein both directly and as a result of activation of ..cap alpha..-adrenergic receptors.

  15. Forskolin inhibits the Gs-stimulated adenylate cyclase in rat ascites hepatoma AH66F cells.

    PubMed

    Miyamoto, K; Sanae, F; Koshiura, R; Matsunaga, T; Hasegawa, T; Takagi, K; Satake, T

    1989-09-01

    Forskolin increased intracellular cyclic AMP and augmented cyclic AMP formation by prostaglandin E1 (PGE1) in normal rat hepatocytes and ascites hepatoma AH66 cells. However, in AH66F cells which were derived from the AH66 cell line, the diterpene only slightly increased the cyclic AMP level, and dose-dependently inhibited the accumulation caused by PGE1. Forskolin dose-dependently activated adenylate cyclase in these membranes, and the magnitude of activation by forskolin was largest in the following order: hepatocytes, AH66 cells, and AH66F cells. This difference may be based on the number of forskolin-binding sites. The binding affinity of forskolin for each cell membrane was similar. The number and affinity of forskolin-binding sites in these cells were not influenced by 5'-guanylylimidodiphosphate [Gpp(NH)p]. In hepatocytes and AH66 cells, forskolin and other adenylate cyclase activators such as PGE1, GTP, Gpp(NH)p, F-, and Mn2+ synergistically increased the enzyme activity. In AH66F cells, the forskolin-stimulated activity was hardly influenced by the GTP analog, and forskolin diminished the activities induced by the GTP analog in a manner similar to that of diterpene alone. Forskolin (10 microM) also significantly inhibited the activities induced by PGE1, GTP, and F-. The effect of forskolin with Mn2+ was additive in AH66F cells. The data suggest that forskolin promotes the interaction between the stimulatory guanine nucleotide-binding protein and the catalytic unit in the membrane of normal hepatocytes and AH66 cells, but it interferes with the coupling in AH66F cells.

  16. Characterization of beta-adrenergic receptors and adenylate cyclase activity in rat brown fat

    SciTech Connect

    Baresi, L.A.; Morley, J.E.; Scarpace, P.J.

    1986-03-01

    Catecholamines stimulate thermogenesis in rat brown fat through a mechanism which involves binding to the beta-adrenergic receptor (BAR), stimulation of adenylate cyclase (AC) and culminating with uncoupling of mitochondrial respiration from ATP synthesis. The authors characterized BAR, AC and cytochrome (cyt) c oxidase in CDF (F-344) interscapular brown fat. Scatchard analysis of (/sup 125/)Iodopindolol binding yields a straight line consistent with a single class of antagonist binding sites with 41.8 +/- 12.0 fmol BAR/mg protein and a K/sub d/ of 118 +/- 15 pM. Binding was both specific and stereospecific. Competition with 1-propranolol (K/sub d/ = 6.7 nM) was 15 times more potent than d-propranolol (K/sub d/ = 103 nM). Competition with isoproterenol (K/sub d/ = 79 nM) was 10 times more potent than epinephrine (K/sub d/ = 820 nM) which was 35 times more potent than norepinephrine (K/sub d/ = 2.9 x 10/sup -5/ M) suggesting predominate beta/sub 2/-type BAR. Cyt c oxidase activity was assessed in brown fat mitochrondrial preparations. The ratio of BAR to cyt c activity was 959 +/- 275 nmol BAR/mol cyc c/min. Isoproterenol (0.1 mM) stimulated AC activity was 24 times GTP (0.1 mM) stimulated AC (98.5 vs 40.7 pmol cAMP/min/mg). NaF-stimulated AC was nine times basal activity (90.5 vs 11.3 pmol cAMP/min/mg). These data demonstrate the presence of a beta-/sub 2/-type BAR coupled to adenylate cyclase in rat brown fat.

  17. Adenyl cyclases and cAMP in plant signaling - past and present.

    PubMed

    Gehring, Chris

    2010-06-25

    In lower eukaryotes and animals 3'-5'-cyclic adenosine monophosphate (cAMP) and adenyl cyclases (ACs), enzymes that catalyse the formation of cAMP from ATP, have long been established as key components and second messengers in many signaling pathways. In contrast, in plants, both the presence and biological role of cAMP have been a matter of ongoing debate and some controversy. Here we shall focus firstly on the discovery of cellular cAMP in plants and evidence for a role of this second messenger in plant signal transduction. Secondly, we shall review current evidence of plant ACs, analyse aspects of their domain organisations and the biological roles of candidate molecules. In addition, we shall assess different approaches based on search motifs consisting of functionally assigned amino acids in the catalytic centre of annotated and/or experimentally tested nucleotide cyclases that can contribute to the identification of novel candidate molecules with AC activity such as F-box and TIR proteins.

  18. Subtyping of Salmonella enterica Subspecies I Using Single-Nucleotide Polymorphisms in Adenylate Cyclase

    PubMed Central

    Abdo, Zaid; Byers, Sara Overstreet; Kriebel, Patrick; Rothrock, Michael J.

    2016-01-01

    Abstract Methods to rapidly identify serotypes of Salmonella enterica subspecies I are of vital importance for protecting the safety of food. To supplement the serotyping method dkgB-linked intergenic sequence ribotyping (ISR), single-nucleotide polymorphisms were characterized within adenylate cyclase (cyaA). The National Center for Biotechnology Information (NCBI) database had 378 cyaA sequences from S. enterica subspecies I, which included 42 unique DNA sequences and 19 different amino acid sequences. Five representative isolates, namely serotypes Typhimurium, Kentucky, Enteritidis phage type PT4, and two variants of Enteritidis phage type PT13a, were differentiated within a microsphere-based fluidics system in cyaA by allele-specific primer extension. Validation against 25 poultry-related environmental Salmonella isolates representing 11 serotypes yielded a ∼89% success rate at identifying the serotype of the isolate, and a different region could be targeted to achieve 100%. When coupled with ISR, all serotypes were differentiated. Phage lineages of serotype Enteritidis 13a and 4 were identified, and a biofilm-forming strain of PT13a was differentiated from a smooth phenotype within phage type. Comparative ranking of mutation indices to genes such as the tRNA transferases, the diguanylate cyclases, and genes used for multilocus sequence typing indicated that cyaA is an appropriate gene for assessing epidemiological trends of Salmonella because of its relative stability in nucleotide composition. PMID:27035032

  19. Mechanism of activation of light-activated phosphodiesterase and evidence for homology with hormone-activated adenylate cyclase

    SciTech Connect

    Bitensky, M.W.; Yamazaki, A.; Wheeler, M.A.; George, J.S.; Rasenick, M.M.

    1983-01-01

    Light-activated cGMP phosphodiesterase (PDE) is one of the effector proteins in the rod outer segments in vertebrate retina. The hydrolysis of cGMP in rod occurs with a speed and light sensitivity which suggests a role for this hydrolysis in visual transduction. In fact, there is electrophysiological data which supports the possibility that cGMP could regulate rod membrane voltage. PDE shows very rapid activation in the presence of photons and GTP. We have called attention to the intriguing analogy between light activated rod phosphodiesterase and hormone activated adenylate cyclase. A number of studies have implicated the binding of GTP to a GTP binding protein as a factor in the hormone dependent activation of adenylate cyclase. Moreover, Cassel and Selinger have shown that hydrolysis of GTP is a component in the inactivation of the hormone dependent adenylate cyclase. We review here recent additional data which provide specific molecular details of the mechanism of light activation of rod PDE as well as demonstrate the exchange of components between light activated PDE and hormone activated cyclase.

  20. 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.; Guillon, G.; Homburger, V.

    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 mass (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.

  1. Guanine nucleotide binding regulatory proteins and adenylate cyclase in livers of streptozotocin- and BB/Wor-diabetic rats. Immunodetection of Gs and Gi with antisera prepared against synthetic peptides.

    PubMed Central

    Lynch, C J; Blackmore, P F; Johnson, E H; Wange, R L; Krone, P K; Exton, J H

    1989-01-01

    Adenylate cyclase in liver plasma membranes from streptozotocin-diabetic (STZ) or BB/Wor spontaneously diabetic rats showed increased responsiveness to GTP, glucagon, fluoroaluminate, and cholera toxin. Basal or forskolin-stimulated activity was unchanged in STZ rats, but increased in BB/Wor rats. No change in the alpha-subunit of Gi (alpha i) was observed in STZ or BB/Wor rats using pertussis toxin-stimulated [32P]ADP-ribosylation. Immunodetection using antibodies against the COOH-terminal decapeptides of alpha T and alpha i-3 showed no change in alpha i in STZ rats and a slight decrease in BB/Wor rats. Angiotensin II inhibition of hepatic adenylate cyclase was not altered in either diabetic rat. In both models of diabetes, Gs alpha-subunits were increased as measured by cholera toxin-stimulated [32P]-ADP-ribosylation of 43-47.5-kD peptides, reconstitution with membranes from S49 cyc- cells or immunoreactivity using antibodies against the COOH-terminal decapeptide of alpha s. These data indicate that STZ-diabetes increases hepatic Gs but does not change Gi or adenylate cyclase catalytic activity. In contrast, BB/Wor rats show increased hepatic Gs and adenylate cyclase. These changes could explain the increase in hepatic cAMP and related dysfunctions observed in diabetes. Images PMID:2498395

  2. Stress-related disorders, pituitary adenylate cyclase-activating peptide (PACAP)ergic system, and sex differences.

    PubMed

    Ramikie, Teniel S; Ressler, Kerry J

    2016-12-01

    Trauma-related disorders, such as posttraumatic stress disorder (PTSD) are remarkably common and debilitating, and are often characterized by dysregulated threat responses. Across numerous epidemiological studies, females have been found to have an approximately twofold increased risk for PTSD and other stress-related disorders. Understanding the biological mechanisms of this differential risk is of critical importance. Recent data suggest that the pituitary adenylate cyclase-activating polypeptide (PACAP) pathway is a critical regulator of the stress response across species. Moreover, increasing evidence suggests that this pathway is regulated by both stress and estrogen modulation and may provide an important window into understanding mechanisms of sex differences in the stress response. We have recently shown that PACAP and its receptor (PAC1R) are critical mediators of abnormal processes after psychological trauma. Notably, in heavily traumatized human subjects, there appears to be a robust sex-specific association of PACAP blood levels and PAC1R gene variants with fear physiology, PTSD diagnosis, and symptoms, specifically in females. The sex-specific association occurs within a single-nucleotide polymorphism (rs2267735) that resides in a putative estrogen response element involved in PAC1R gene regulation. Complementing these human data, the PAC1R messenger RNA is induced with fear conditioning or estrogen replacement in rodent models. These data suggest that perturbations in the PACAP-PAC1R pathway are regulated by estrogen and are involved in abnormal fear responses underlying PTSD.

  3. Development of a novel photoreactive calmodulin derivative: Cross-linking of purified adenylate cyclase from bovine brain

    SciTech Connect

    Harrison, J.K.; Lawton, R.G.; Gnegy, M.E. )

    1989-07-11

    A novel photoreactive calmodulin (CaM) derivative was developed and used to label the purified CaM-sensitive adenylate cyclase from bovine cortex. {sup 125}I-CaM was conjugated with the heterobifunctional cross-linking agent p-nitrophenyl 3-diazopyruvate (DAPpNP). Spectral data indicated that diazopyruvoyl (DAP) groups were incorporated into the CaM molecule. Iodo-CaM-DAPs behaved like native CaM with respect to (1) Ca{sup 2+}-dependent enhanced mobility on sodium dodecyl sulfate-polyacrylamide gels and (2) Ca{sup 2+}-dependent stimulation of adenylate cyclase activity. {sup 125}I-CaM-DAP photochemically cross-linked to CaM-binding proteins in a manner that was both Ca{sup 2+} dependent and CaM specific. Photolysis of forskolin-agarose-purified adenylate cyclase from bovine cortex with {sup 125}I-CaM-DAP produced a single cross-linked product which migrates on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of approximately 140,000.

  4. 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/mg 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.

  5. Studies of the cAMP mediated aggregation in Dictyostelium discoideum: receptor mediated activation of the adenylate cyclase

    SciTech Connect

    Theibert, W.E.A.B.

    1985-01-01

    Dictyostelium discoideum, a eukaryotic amoeba of the cellular slime mold family, provides an interesting paradigm in developmental biology. During development, hundreds of thousands of cells aggregate to form a multicellular aggregate. Aggregation is mediated by chemotaxis and chemical signaling. Waves of adenosine 3'-5' cyclic monophosphate (cAMP) propagate through the monolayer and provide transient gradients for chemotaxis. The author has used a reversible inhibitor of the cAMP signaling response to demonstrate that adaptation to cAMP is independent of the activation of the adenylate cyclase and therefore is not caused by the rise in intracellular cAMP. Next, it is shown that adenosine inhibits the cAMP signaling response. Inhibition is rapid, reversible, and depends on the cAMP stimulus concentration. Then the specificity of the cAMP receptors which mediates signaling is determined and compared with the receptors which mediate chemotaxis, the cGMP response, and cAMP binding antagonism. The cAMP surface receptor has been identified by photoaffinity labeling intact cells with (/sup 32/P)-8-N/sub 3/-cAMP using an ammonium sulfate binding stabilization technique. The photoactivated ligand specifically labels a polypeptide, localized to the membrane fraction, which migrates as a closely spaced doublet on SDS Page.

  6. [Structure, localization and physiologic role of pituitary adenylate cyclase activating polypeptide (PACAP)].

    PubMed

    Vincze, E; Köves, K

    2001-03-11

    PACAP was isolated on the basis of its ability to stimulate adenylate cyclase in primary anterior pituitary cell culture from ovine hypothalami by Miyata et al. in 1989. This peptide is structurally related to the secretin family and shows a 67% sequence homology with vasoactive intestinal polypeptide (VIP). The amino acid sequence of PACAP has been highly preserved during the evolution that may be connected with its important physiological role. Similar to other "brain-gut peptides" PACAP is localized not only in the central but in the peripheral nervous system and in non-neural tissues as well. In addition to its hypophysiotropic effects in the hypothalamo-hypophysial system PACAP exerts its effects on water-salt balance, cardiovascular functions, gastrointestinal motility and secretion and also on the regulation of reproductive functions. PACAP has a role in certain neuro-immuno-endocrine processes, in the differentiation of the nervous system, and it has neuroprotective effects in the case of ischaemia and various toxic agents. Locally PACAP takes its effects as an auto- and paracrine hormone, a neurotransmitter or a neuromodulator in different organs. Besides VIP, PACAP plays an important role in the function of the photo-neuro-endocrine system.

  7. Adenylate Cyclase Type III Is Not a Ubiquitous Marker for All Primary Cilia during Development

    PubMed Central

    Antal, Maria Cristina; Bénardais, Karelle; Samama, Brigitte; Auger, Cyril; Schini-Kerth, Valérie; Ghandour, Said; Boehm, Nelly

    2017-01-01

    Adenylate cyclase type III (AC3) is localized in plasma membrane of neuronal primary cilium and can be used as a marker of this cilium. AC3 has also been detected in some other primary cilia such as those of fibroblasts, synoviocytes or astrocytes. Despite the presence of a cilium in almost all cell types, we show that AC3 is not a common marker of all primary cilia of different human and mouse tissues during development. In peripheral organs, AC3 is present mainly in primary cilia in cells of the mesenchymal lineage (fibroblasts, chondroblasts, osteoblasts-osteocytes, odontoblasts, muscle cells and endothelial cells). In epithelia, the apical cilium of renal and pancreatic tubules and of ductal plate in liver is AC3-negative whereas the cilium of basal cells of stratified epithelia is AC3-positive. Using fibroblasts cell culture, we show that AC3 appears at the plasma membrane of the primary cilium as soon as this organelle develops. The functional significance of AC3 localization at the cilium membrane in some cells but not others has to be investigated in relationship with cell physiology and expression at the cilium plasma membrane of specific upstream receptors. PMID:28122017

  8. Bordetella pertussis adenylate cyclase: purification and characterization of the toxic form of the enzyme.

    PubMed Central

    Rogel, A; Schultz, J E; Brownlie, R M; Coote, J G; Parton, R; Hanski, E

    1989-01-01

    Bordetella pertussis produces a calmodulin-sensitive adenylate cyclase (AC) which is an essential virulence factor in mammalian pertussis. Here we report the purification and characterization of the toxic form of the enzyme, which penetrates eukaryotic cells and generates high levels of intracellular cAMP. This form was purified from an extract of B.pertussis strain carrying a recombinant plasmid which over-produced both enzymatic and toxic activities of the enzyme. Western blot analysis of the extract using anti-B.pertussis AC antibodies detected only one protein of 200 kd. However, gel filtration of the extract resolved two peaks of enzymatic activity. The first peak of aggregated material contained greater than 70% of the total enzymatic activity, and the second peak contained the majority of the toxic activity. Purification of the enzyme from both peaks yielded proteins of 200 kd, with similar biochemical and immunological properties. Yet only the enzyme purified from the second peak could penetrate human lymphocyte and catalyse the formation of intracellular cAMP. B.pertussis AC gene expressed in Escherichia coli produced a calmodulin-dependent enzyme of 200 kd, which lacked lymphocyte penetration capacity. It is proposed that a post-translational modification that occurs in B.pertussis but not in E.coli confers upon the 200 kd protein of B.pertussis AC the toxic properties. Images PMID:2555185

  9. 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.

  10. Adenylate cyclase-associated protein 1 overexpressed in pancreatic cancers is involved in cancer cell motility.

    PubMed

    Yamazaki, Ken; Takamura, Masaaki; Masugi, Yohei; Mori, Taisuke; Du, Wenlin; Hibi, Taizo; Hiraoka, Nobuyoshi; Ohta, Tsutomu; Ohki, Misao; Hirohashi, Setsuo; Sakamoto, Michiie

    2009-04-01

    Pancreatic cancer has the worst prognosis among cancers due to the difficulty of early diagnosis and its aggressive behavior. To characterize the aggressiveness of pancreatic cancers on gene expression, pancreatic cancer xenografts transplanted into severe combined immunodeficient mice served as a panel for gene-expression profiling. As a result of profiling, the adenylate cyclase-associated protein 1 (CAP1) gene was shown to be overexpressed in all of the xenografts. The expression of CAP1 protein in all 73 cases of pancreatic cancer was recognized by immunohistochemical analyses. The ratio of CAP1-positive tumor cells in clinical specimens was correlated with the presence of lymph node metastasis and neural invasion, and also with the poor prognosis of patients. Immunocytochemical analyses in pancreatic cancer cells demonstrated that CAP1 colocalized to the leading edge of lamellipodia with actin. Knockdown of CAP1 by RNA interference resulted in the reduction of lamellipodium formation, motility, and invasion of pancreatic cancer cells. This is the first report demonstrating the overexpression of CAP1 in pancreatic cancers and suggesting the involvement of CAP1 in the aggressive behavior of pancreatic cancer cells.

  11. Pituitary Adenylate Cyclase-Activating Polypeptide Reverses Ammonium Metavanadate-Induced Airway Hyperresponsiveness in Rats

    PubMed Central

    Tlili, Mounira; Rouatbi, Sonia; Sriha, Badreddine; Ben Rhouma, Khémais; Sakly, Mohsen; Vaudry, David; Wurtz, Olivier; Tebourbi, Olfa

    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

  12. Adenyl cyclase activator forskolin protects against Huntington's disease-like neurodegenerative disorders

    PubMed Central

    Mehan, Sidharth; Parveen, Shaba; Kalra, Sanjeev

    2017-01-01

    Long term suppression of succinate dehydrogenase by selective inhibitor 3-nitropropionic acid has been used in rodents to model Huntington's disease where mitochondrial dysfunction and oxidative damages are primary pathological hallmarks for neuronal damage. Improvements in learning and memory abilities, recovery of energy levels, and reduction of excitotoxicity damage can be achieved through activation of Adenyl cyclase enzyme by a specific phytochemical forskolin. In this study, intraperitoneal administration of 10 mg/kg 3-nitropropionic acid for 15 days in rats notably reduced body weight, worsened motor cocordination (grip strength, beam crossing task, locomotor activity), resulted in learning and memory deficits, greatly increased acetylcholinesterase, lactate dehydrogenase, nitrite, and malondialdehyde levels, obviously decreased adenosine triphosphate, succinate dehydrogenase, superoxide dismutase, catalase, and reduced glutathione levels in the striatum, cortex and hippocampus. Intragastric administration of forskolin at 10, 20, 30 mg/kg dose-dependently reversed these behavioral, biochemical and pathological changes caused by 3-nitropropionic acid. These results suggest that forskolin exhibits neuroprotective effects on 3-nitropropionic acid-induced Huntington's disease-like neurodegeneration.

  13. Adenylate cyclase A acting on PKA mediates induction of stalk formation by cyclic diguanylate at the Dictyostelium organizer

    PubMed Central

    Chen, Zhi-Hui; Singh, Reema; Cole, Christian; Lawal, Hajara Mohammed; Schilde, Christina; Febrer, Melanie; Barton, Geoffrey J.; Schaap, Pauline

    2017-01-01

    Coordination of cell movement with cell differentiation is a major feat of embryonic development. The Dictyostelium stalk always forms at the organizing tip, by a mechanism that is not understood. We previously reported that cyclic diguanylate (c-di-GMP), synthesized by diguanylate cyclase A (DgcA), induces stalk formation. Here we used transcriptional profiling of dgca− structures to identify target genes for c-di-GMP, and used these genes to investigate the c-di-GMP signal transduction pathway. We found that knockdown of cAMP-dependent protein kinase (PKA) activity in prestalk cells reduced stalk gene induction by c-di-GMP, whereas PKA activation bypassed the c-di-GMP requirement for stalk gene expression. c-di-GMP caused a persistent increase in cAMP, which still occurred in mutants lacking the adenylate cyclases ACG or ACR, or the cAMP phosphodiesterase RegA. However, both inhibition of adenylate cyclase A (ACA) with SQ22536 and incubation of a temperature-sensitive ACA mutant at the restrictive temperature prevented c-di-GMP–induced cAMP synthesis as well as c-di-GMP–induced stalk gene transcription. ACA produces the cAMP pulses that coordinate Dictyostelium morphogenetic cell movement and is highly expressed at the organizing tip. The stalk-less dgca− mutant regained its stalk by expression of a light-activated adenylate cyclase from the ACA promoter and exposure to light, indicating that cAMP is also the intermediate for c-di-GMP in vivo. Our data show that the more widely expressed DgcA activates tip-expressed ACA, which then acts on PKA to induce stalk genes. These results explain why stalk formation in Dictyostelia always initiates at the site of the morphogenetic organizer. PMID:28057864

  14. Plant-activated bacterial receptor adenylate cyclases modulate epidermal infection in the Sinorhizobium meliloti-Medicago symbiosis.

    PubMed

    Tian, Chang Fu; Garnerone, Anne-Marie; Mathieu-Demazière, Céline; Masson-Boivin, Catherine; Batut, Jacques

    2012-04-24

    Legumes and soil bacteria called rhizobia have coevolved a facultative nitrogen-fixing symbiosis. Establishment of the symbiosis requires bacterial entry via root hair infection threads and, in parallel, organogenesis of nodules that subsequently are invaded by bacteria. Tight control of nodulation and infection is required to maintain the mutualistic character of the interaction. Available evidence supports a passive bacterial role in nodulation and infection after the microsymbiont has triggered the symbiotic plant developmental program. Here we identify in Sinorhizobium meliloti, the Medicago symbiont, a cAMP-signaling regulatory cascade consisting of three receptor-like adenylate cyclases, a Crp-like regulator, and a target gene of unknown function. The cascade is activated specifically by a plant signal during nodule organogenesis. Cascade inactivation results in a hyperinfection phenotype consisting of abortive epidermal infection events uncoupled from nodulation. These findings show that, in response to a plant signal, rhizobia play an active role in the control of infection. We suggest that rhizobia may modulate the plant's susceptibility to infection. This regulatory loop likely aims at optimizing legume infection.

  15. Characterization of the norepinephrine-activation of adenylate cyclase suggests a role in memory affirmation pathways. Overexposure to epinephrine inactivates adenylate cyclase, a causal pathway for stress-pathologies.

    PubMed

    Bennun, Alfred

    2010-05-01

    Incubation with noradrenaline (norepinephrine) of isolated membranes of rat's brain corpus striatum and cortex, showed that ionic-magnesium (Mg(2+)) is required for the neurotransmitter activatory response of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing) (EC 4.6.1.1)], AC. An Mg(2+)-dependent response to the activatory effects of adrenaline, and subsequent inhibition by calcium, suggest capability for a turnover, associated with cyclic changes in membrane potential and participation in a short-term memory pathway. In the cell, the neurotransmitter by activating AC generates intracellular cyclic AMP. Calcium entrance in the cell inhibits the enzyme. The increment of cyclic AMP activates kinase A and their protein phosphorylating activity, allowing a long-term memory pathway. Hence, consolidating neuronal circuits, related to emotional learning and memory affirmation. The activatory effect relates to an enzyme-noradrenaline complex which may participate in the physiology of the fight or flight response, by prolonged exposure. However, the persistence of an unstable enzyme complex turns the enzyme inactive. Effect concordant, with the observation that prolonged exposure to adrenaline, participates in the etiology of stress triggered pathologies. At the cell physiological level AC responsiveness to hormones could be modulated by the concentration of chelating metabolites. These ones produce the release of free ATP(4-), a negative modulator of AC and the Mg(2+) activated insulin receptor tyrosine kinase (IRTK), thus, allowing an integration of the hormonal response of both enzymes by ionic controls. This effect could supersede the metabolic feedback control by energy charge. Accordingly, maximum hormonal response of both enzymes, to high Mg(2+) and low free ATP(4-), allows a correlation with the known effects of low caloric intake increasing average life expectancy.

  16. D-1 dopaminergic and beta-adrenergic stimulation of adenylate cyclase in a clone derived from the human astrocytoma cell line G-CCM.

    PubMed

    Balmforth, A J; Ball, S G; Freshney, R I; Graham, D I; McNamee, H B; Vaughan, P F

    1986-09-01

    Clones have been isolated from the human astrocytoma cell line G-CCM. Homogenates of clone D384 contain an adenylate cyclase that is stimulated by 3,4-dihydroxyphenylethylamine (dopamine), noradrenaline, and isoprenaline with Ka apparent values of 4, 56, and 2.7 microM, respectively. The Ka apparent value for dopamine was increased by the D-1 antagonist cis-flupenthixol, 25 and 100 nM, to 23 and 190 microM, respectively, but was unaffected by propranolol (1 microM). Noradrenaline stimulation of adenylate cyclase was only partially inhibited by either propranolol (10 microM) or cis-flupenthixol (1 microM). Propranolol (10 microM), but not cis-flupenthixol (1 microM), prevented stimulation by isoprenaline. The stimulation of adenylate cyclase by dopamine and noradrenaline remained unchanged in the presence of phentolamine (1 microM) and sulpiride (1 microM). These results suggest that clone D384 contains both D-1 dopaminergic and beta-adrenergic receptors coupled to adenylate cyclase. Dopamine stimulates D384 adenylate cyclase through D-1 receptors, isoprenaline via beta-receptors, and noradrenaline through both receptors.

  17. Heterologous desensitization of adenylate cyclase from pigeon erythrocytes under the action of the catalytic subunit of cAMP-dependent protein kinase

    SciTech Connect

    Popov, K.M.; Bulargina, T.V.; Severin, E.S.

    1985-09-20

    Preincubation of the plasma membranes from pigeon erythrocytes with the catalytic subunit of cAMP-dependent protein kinase leads to desensitization of adenylate cyclase of the erythrocytes. The adenylate cyclase activity, measured in the presence of 10 ..mu..M isoproterenol and 50 ..mu..M GTP-..gamma..-S, is decreased by 40% in 10 min of incubation, while the activity in the presence of 50 ..mu..M GTP-..gamma..-S is decreased by 35% in 20 min. The decrease in the adenylate cyclase activity is due to an increase in the lag phase of activation of the enzyme in the presence of a GTP analog stable to hydrolysis and a decrease in the activity in the steady-state phase of activation. Heterologous desensitization of adenylate cyclase under the action of cAMP-dependent protein kinase is coupled with a decrease in the number of ..beta..-adrenoreceptors capable of passing into a state of high affinity for antagonists in the absence of guanylic nucleotides. The influence of the catalytic subunit on adenylate cyclase entirely models the process of desensitization of the enzyme absorbed in the influence of isoproterenol or cAMP on erythrocytes.

  18. Inhibition of adenylate cyclase attenuates muscarinic Ca²(+) signaling by a PKA-independent mechanism in rat carotid body Type I cells.

    PubMed

    Thompson, Carrie M; Wyatt, Christopher N

    2011-01-31

    Carotid body (CB) Type I cells respond to hypoxia by releasing excitatory and inhibitory neurotransmitters. This mechanism leads to increased firing of the carotid sinus nerve (CSN) which alters breathing to maintain blood gases within the physiological range. Acetylcholine targets both muscarinic and nicotinic receptors in the rat CB, acting postsynaptically on CSN and presynaptically on Type I cells. Muscarinic Ca²(+) signaling is inhibited by the activation of G(i)-coupled receptors including histamine H3 receptors. Here inhibition of adenylate cyclase with SQ22536 mimicked H3 receptor activation. Using Ca²(+) imaging techniques it was observed that inhibition of muscarinic Ca²(+) signaling was independent of protein kinase A (PKA) as PKA inhibitors H89 and KT5720 were without effect on the muscarinic Ca²(+) response. By contrast the Epac (exchange protein activated by cAMP) inhibitor brefeldin A inhibited muscarinic Ca²(+) signaling whereas the Epac activator 8-pCPT-2'-O-Me-cAMP-AM potentiated Ca²(+) signaling. Thus in Type I cells inhibition of adenylate cyclase inhibited muscarinic Ca²(+) signaling via a PKA-independent pathway that may rely upon modulation of Epac.

  19. Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer

    PubMed Central

    Veneziano, Rémi; Rossi, Claire; Chenal, Alexandre; Devoisselle, Jean-Marie; Ladant, Daniel; Chopineau, Joel

    2013-01-01

    Numerous bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, where they exert their cytotoxic effects. Our model toxin, the adenylate cyclase (CyaA) from Bordetella pertussis, is able to invade eukaryotic cells by translocating its catalytic domain directly across the plasma membrane of target cells. To characterize its original translocation process, we designed an in vitro assay based on a biomimetic membrane model in which a tethered lipid bilayer (tBLM) is assembled on an amine-gold surface derivatized with calmodulin (CaM). The assembled bilayer forms a continuous and protein-impermeable boundary completely separating the underlying calmodulin (trans side) from the medium above (cis side). The binding of CyaA to the tBLM is monitored by surface plasmon resonance (SPR) spectroscopy. CyaA binding to the immobilized CaM, revealed by enzymatic activity, serves as a highly sensitive reporter of toxin translocation across the bilayer. Translocation of the CyaA catalytic domain was found to be strictly dependent on the presence of calcium and also on the application of a negative potential, as shown earlier in eukaryotic cells. Thus, CyaA is able to deliver its catalytic domain across a biological membrane without the need for any eukaryotic components besides CaM. This suggests that the calcium-dependent CyaA translocation may be driven in part by the electrical field across the membrane. This study’s in vitro demonstration of toxin translocation across a tBLM provides an opportunity to explore the molecular mechanisms of protein translocation across biological membranes in precisely defined experimental conditions. PMID:24297899

  20. Pituitary Adenylate-Cyclase Activating Polypeptide Regulates Hunger- and Palatability-Induced Binge Eating

    PubMed Central

    Hurley, Matthew M.; Maunze, Brian; Block, Megan E.; Frenkel, Mogen M.; Reilly, Michael J.; Kim, Eugene; Chen, Yao; Li, Yan; Baker, David A.; Liu, Qing-Song; Choi, SuJean

    2016-01-01

    While pituitary adenylate cyclase activating polypeptide (PACAP) signaling in the hypothalamic ventromedial nuclei (VMN) has been shown to regulate feeding, a challenge in unmasking a role for this peptide in obesity is that excess feeding can involve numerous mechanisms including homeostatic (hunger) and hedonic-related (palatability) drives. In these studies, we first isolated distinct feeding drives by developing a novel model of binge behavior in which homeostatic-driven feeding was temporally separated from feeding driven by food palatability. We found that stimulation of the VMN, achieved by local microinjections of AMPA, decreased standard chow consumption in food-restricted rats (e.g., homeostatic feeding); surprisingly, this manipulation failed to alter palatable food consumption in satiated rats (e.g., hedonic feeding). In contrast, inhibition of the nucleus accumbens (NAc), through local microinjections of GABA receptor agonists baclofen and muscimol, decreased hedonic feeding without altering homeostatic feeding. PACAP microinjections produced the site-specific changes in synaptic transmission needed to decrease feeding via VMN or NAc circuitry. PACAP into the NAc mimicked the actions of GABA agonists by reducing hedonic feeding without altering homeostatic feeding. In contrast, PACAP into the VMN mimicked the actions of AMPA by decreasing homeostatic feeding without affecting hedonic feeding. Slice electrophysiology recordings verified PACAP excitation of VMN neurons and inhibition of NAc neurons. These data suggest that the VMN and NAc regulate distinct circuits giving rise to unique feeding drives, but that both can be regulated by the neuropeptide PACAP to potentially curb excessive eating stemming from either drive. PMID:27597817

  1. Neurally released pituitary adenylate cyclase-activating polypeptide enhances guinea pig intrinsic cardiac neurone excitability.

    PubMed

    Tompkins, John D; Ardell, Jeffrey L; Hoover, Donald B; Parsons, Rodney L

    2007-07-01

    Intracellular recordings were made in vitro from guinea-pig cardiac ganglia to determine whether endogenous neuropeptides such as pituitary adenylate cyclase-activating polypeptide (PACAP) or substance P released during tetanic neural stimulation modulate cardiac neurone excitability and/or contribute to slow excitatory postsynaptic potentials (sEPSPs). When nicotinic and muscarinic receptors were blocked by hexamethonium and atropine, 20 Hz stimulation for 10 s initiated a sEPSP in all innervated neurones. In 40% of the cells, excitability was enhanced after termination of the sEPSP. This suggested that non-cholinergic receptor-mediated mechanisms contributed to the sEPSP and modulated neuronal excitability. Exogenous PACAP and substance P initiated a slow depolarization in the neurones whereas neuronal excitability was only increased by PACAP. When ganglia were treated with the PAC1 antagonist PACAP6-38 (500 nM), the sEPSP evoked by 20 Hz stimulation was reduced by approximately 50% and an enhanced excitability occurred in only 10% of the cells. These observations suggested that PACAP released from preganglionic nerve terminals during tetanic stimulation enhanced neuronal excitability and evoked sEPSPs. After addition of 1 nM PACAP to the bath, 7 of 9 neurones exhibited a tonic firing pattern whereas in untreated preparations, the neurons had a phasic firing pattern. PACAP6-38 (500 nM) diminished the increase in excitability caused by 1 nM PACAP so that only 4 of 13 neurones exhibited a tonic firing pattern and the other 9 cells retained a phasic firing pattern. These findings indicate that PACAP can be released by tetanic neural stimulation in vitro and increase the excitability of intrinsic cardiac neurones. We hypothesize that in vivo PACAP released during preganglionic firing may modulate neurotransmission within the intrinsic cardiac ganglia.

  2. Role of phosphodiesterase and adenylate cyclase isozymes in murine colonic glucagon-like peptide 1 secreting cells

    PubMed Central

    Friedlander, Ronn S; Moss, Catherine E; Mace, Jessica; Parker, Helen E; Tolhurst, Gwen; Habib, Abdella M; Wachten, Sebastian; Cooper, Dermot M; Gribble, Fiona M; Reimann, Frank

    2011-01-01

    BACKGROUND AND PURPOSE Glucagon-like peptide-1 (GLP-1) is secreted from enteroendocrine L-cells after food intake. Increasing GLP-1 signalling either through inhibition of the GLP-1 degrading enzyme dipeptidyl-peptidase IV or injection of GLP-1-mimetics has recently been successfully introduced for the treatment of type 2 diabetes. Boosting secretion from the L-cell has so far not been exploited, due to our incomplete understanding of L-cell physiology. Elevation of cyclic adenosine monophosphate (cAMP) has been shown to be a strong stimulus for GLP-1 secretion and here we investigate the activities of adenylate cyclase (AC) and phosphodiesterase (PDE) isozymes likely to shape cAMP responses in L-cells. EXPERIMENTAL APPROACH Expression of AC and PDE isoforms was quantified by RT-PCR. Single cell responses to stimulation or inhibition of AC and PDE isoforms were monitored with real-time cAMP probes. GLP-1 secretion was assessed by elisa. KEY RESULTS Quantitative PCR identified expression of protein kinase C- and Ca2+-activated ACs, corresponding with phorbolester and cytosolic Ca2+-stimulated cAMP elevation. Inhibition of PDE2, 3 and 4 were found to stimulate GLP-1 secretion from murine L-cells in primary culture. This corresponded with cAMP elevations monitored with a plasma membrane targeted cAMP probe. Inhibition of PDE3 but not PDE2 was further shown to prevent GLP-1 secretion in response to guanylin, a peptide secreted into the gut lumen, which had not previously been implicated in L-cell secretion. CONCLUSIONS AND IMPLICATIONS Our results reveal several mechanisms shaping cAMP responses in GLP-1 secreting cells, with some of the molecular components specifically expressed in L-cells when compared with their epithelial neighbours, thus opening new strategies for targeting these cells therapeutically. PMID:21054345

  3. Mutation in the β-hairpin of the Bordetella pertussis adenylate cyclase toxin modulates N-lobe conformation in calmodulin

    SciTech Connect

    Springer, Tzvia I.; Goebel, Erich; Hariraju, Dinesh; Finley, Natosha L.

    2014-10-10

    Highlights: • Bordetella pertussis adenylate cyclase toxin modulates bi-lobal structure of CaM. • The structure and stability of the complex rely on intermolecular associations. • A novel mode of CaM-dependent activation of the adenylate cyclase toxin is proposed. - Abstract: Bordetella pertussis, causative agent of whooping cough, produces an adenylate cyclase toxin (CyaA) that is an important virulence factor. In the host cell, the adenylate cyclase domain of CyaA (CyaA-ACD) is activated upon association with calmodulin (CaM), an EF-hand protein comprised of N- and C-lobes (N-CaM and C-CaM, respectively) connected by a flexible tether. Maximal CyaA-ACD activation is achieved through its binding to both lobes of intact CaM, but the structural mechanisms remain unclear. No high-resolution structure of the intact CaM/CyaA-ACD complex is available, but crystal structures of isolated C-CaM bound to CyaA-ACD shed light on the molecular mechanism by which this lobe activates the toxin. Previous studies using molecular modeling, biochemical, and biophysical experiments demonstrate that CyaA-ACD’s β-hairpin participates in site-specific interactions with N-CaM. In this study, we utilize nuclear magnetic resonance (NMR) spectroscopy to probe the molecular association between intact CaM and CyaA-ACD. Our results indicate binding of CyaA-ACD to CaM induces large conformational perturbations mapping to C-CaM, while substantially smaller structural changes are localized primarily to helices I, II, and IV, and the metal-binding sites in N-CaM. Site-specific mutations in CyaA-ACD’s β-hairpin structurally modulate N-CaM, resulting in conformational perturbations in metal binding sites I and II, while no significant structural modifications are observed in C-CaM. Moreover, dynamic light scattering (DLS) analysis reveals that mutation of the β-hairpin results in a decreased hydrodynamic radius (R{sub h}) and reduced thermal stability in the mutant complex. Taken

  4. Allosteric equilibrium model explains steady-state coupling of beta-adrenergic receptors to adenylate cyclase in turkey erythrocyte membranes.

    PubMed

    Ugur, O; Onaran, H O

    1997-05-01

    We used a simple experimental approach to clarify some contradictory predictions of the collision coupling and equilibrium models (e.g. ternary complex, two-state ternary complex or quinternary complex), which describe G-protein-mediated beta-adrenergic receptor signalling in essentially different manners. Analysis of the steady-state coupling of beta-adrenoceptors to adenylate cyclase in turkey erythrocyte membranes showed that: (1) in the absence of an agonist, Gpp(NH)p (a hydrolysis-resistant analogue of GTP) can activate adenylate cyclase very slowly; (2) this activity reaches a steady state in approx. 5 h, the extent of activity depending on the concentration of the nucleotide; (3) isoprenaline-activated steady-state adenylate cyclase can be inactivated by propranolol (a competitive antagonist that relaxes the receptor activation), in the presence of Gpp(NH)p (which provides a virtual absence of GTPase) and millimolar concentrations of Mg2+ (the rate of this inactivation is relatively fast); (4) increasing the concentration of Gpp(NH)p can saturate the steady-state activity of adenylate cyclase. The saturated enzyme activity was lower than that induced by isoprenaline under the same conditions. This additional agonist-induced activation was reversible. In the light of these results, we conclude that agonist can also activate the guanine nucleotide-saturated system in the absence of GTPase by a mechanism other than guanine nucleotide exchange. We explain these phenomena in the framework of a quinternary complex model as an agonist-induced and receptor-mediated dissociation of guanine nucleotide-saturated residual heterotrimer, the equilibrium concentration of which is not necessarily zero. These results, which suggest a continuous interaction between receptor and G-protein, can hardly be accommodated by the collision coupling model that was originally suggested for the present experimental system and then applied to many other G-protein systems. Therefore we

  5. Part II: Biochemical changes after pituitary adenylate cyclase-activating polypeptide-38 infusion in migraine patients.

    PubMed

    Guo, Song; Vollesen, Anne Luise Haulund; Hansen, Young Bae Lee; Frandsen, Erik; Andersen, Malene Rohr; Amin, Faisal Mohammad; Fahrenkrug, Jan; Olesen, Jes; Ashina, Messoud

    2017-02-01

    Background Intravenous infusion of pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) provokes migraine attacks in 65-70% of migraine without aura (MO) patients. We investigated whether PACAP38 infusion causes changes in the endogenous production of PACAP38, vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), tumour necrosis factor alpha (TNFα), S100 calcium binding protein B (S100B), neuron-specific enolase and pituitary hormones in migraine patients. Methods We allocated 32 previously genotyped MO patients to receive intravenous infusion PACAP38 (10 pmol/kg/minute) for 20 minutes and recorded migraine-like attacks. Sixteen of the patients were carriers of the risk allele rs2274316 ( MEF2D), which confers increased risk of MO and may regulate PACAP38 expression, and 16 were non-carriers. We collected blood samples at baseline and 20, 30, 40, 60 and 90 minutes after the start of the infusion. A control group of six healthy volunteers received intravenous saline. Results PACAP38 infusion caused significant changes in plasma concentrations of VIP ( p = 0.026), prolactin ( p = 0.011), S100B ( p < 0.001) and thyroid-stimulating hormone (TSH; p = 0.015), but not CGRP ( p = 0.642) and TNFα ( p = 0.535). We found no difference in measured biochemical variables after PACAP38 infusion in patients who later developed migraine-like attacks compared to those who did not ( p > 0.05). There was no difference in the changes of biochemical variables between patients with and without the MEF2D-associated gene variant ( p > 0.05). Conclusion PACAP38 infusion elevated the plasma levels of VIP, prolactin, S100B and TSH, but not CGRP and TNFα. Development of delayed migraine-like attacks or the presence of the MEF2D gene variant was not associated with pre-ictal changes in plasma levels of neuropeptides, TNFα and pituitary hormones.

  6. Vibrio vulnificus Biotype 3 Multifunctional Autoprocessing RTX Toxin Is an Adenylate Cyclase Toxin Essential for Virulence in Mice

    PubMed Central

    Ziolo, Kevin J.; Jeong, Hee-Gon; Kwak, Jayme S.; Yang, Shuangni; Lavker, Robert M.

    2014-01-01

    Vibrio vulnificus is an environmental organism that causes both food-borne and wound infections with high morbidity and mortality in humans. The annual incidence and global distribution of infections associated with this pathogen are increasing with climate change. In the late 1990s, an outbreak of tilapia-associated wound infections in Israel was linked to a previously unrecognized variant of V. vulnificus designated biotype 3. The sudden emergence and clonality of the outbreak suggest that this strain may be a true newly emergent pathogen with novel virulence properties compared to those of other V. vulnificus strains. In a subcutaneous infection model to mimic wound infection, the multifunctional autoprocessing RTX (MARTX) toxin of biotype 3 strains was shown to be an essential virulence factor contributing to highly inflammatory skin wounds with severe damage affecting every tissue layer. We conducted a sequencing-based analysis of the MARTX toxin and found that biotype 3 MARTX toxin has an effector domain structure distinct from that of either biotype 1 or biotype 2. Of the two new domains identified, a domain similar to Pseudomonas aeruginosa ExoY was shown to confer adenylate cyclase activity on the MARTX toxin. This is the first demonstration that the biotype 3 MARTX toxin is essential for virulence and that the ExoY-like MARTX effector domain is a catalytically active adenylate cyclase. PMID:24614656

  7. Vibrio vulnificus biotype 3 multifunctional autoprocessing RTX toxin is an adenylate cyclase toxin essential for virulence in mice.

    PubMed

    Ziolo, Kevin J; Jeong, Hee-Gon; Kwak, Jayme S; Yang, Shuangni; Lavker, Robert M; Satchell, Karla J F

    2014-05-01

    Vibrio vulnificus is an environmental organism that causes both food-borne and wound infections with high morbidity and mortality in humans. The annual incidence and global distribution of infections associated with this pathogen are increasing with climate change. In the late 1990s, an outbreak of tilapia-associated wound infections in Israel was linked to a previously unrecognized variant of V. vulnificus designated biotype 3. The sudden emergence and clonality of the outbreak suggest that this strain may be a true newly emergent pathogen with novel virulence properties compared to those of other V. vulnificus strains. In a subcutaneous infection model to mimic wound infection, the multifunctional autoprocessing RTX (MARTX) toxin of biotype 3 strains was shown to be an essential virulence factor contributing to highly inflammatory skin wounds with severe damage affecting every tissue layer. We conducted a sequencing-based analysis of the MARTX toxin and found that biotype 3 MARTX toxin has an effector domain structure distinct from that of either biotype 1 or biotype 2. Of the two new domains identified, a domain similar to Pseudomonas aeruginosa ExoY was shown to confer adenylate cyclase activity on the MARTX toxin. This is the first demonstration that the biotype 3 MARTX toxin is essential for virulence and that the ExoY-like MARTX effector domain is a catalytically active adenylate cyclase.

  8. Vasoactive intestinal polypeptide requires parallel changes in adenylate cyclase and phospholipase C to entrain circadian rhythms to a predictable phase

    PubMed Central

    An, Sungwon; Irwin, Robert P.; Allen, Charles N.; Tsai, Connie

    2011-01-01

    Circadian oscillations in the suprachiasmatic nucleus (SCN) depend on transcriptional repression by Period (PER)1 and PER2 proteins within single cells and on vasoactive intestinal polypeptide (VIP) signaling between cells. Because VIP is released by SCN neurons in a circadian pattern, and, after photic stimulation, it has been suggested to play a role in the synchronization to environmental light cycles. It is not known, however, if or how VIP entrains circadian gene expression or behavior. Here, we tested candidate signaling pathways required for VIP-mediated entrainment of SCN rhythms. We found that single applications of VIP reset PER2 rhythms in a time- and dose-dependent manner that differed from light. Unlike VIP-mediated signaling in other cell types, simultaneous antagonism of adenylate cyclase and phospholipase C activities was required to block the VIP-induced phase shifts of SCN rhythms. Consistent with this, VIP rapidly increased intracellular cAMP in most SCN neurons. Critically, daily VIP treatment entrained PER2 rhythms to a predicted phase angle within several days, depending on the concentration of VIP and the interval between VIP applications. We conclude that VIP entrains circadian timing among SCN neurons through rapid and parallel changes in adenylate cyclase and phospholipase C activities. PMID:21389307

  9. Evidence for a presynaptic adenylate cyclase system facilitating (TH)norepinephrine release from rat brain neocortex slices and synaptosomes

    SciTech Connect

    Schoffelmeer, A.N.; Hogenboom, F.; Mulder, A.H.

    1985-10-01

    The effects of drugs known to enhance intracellular cyclic AMP levels on depolarization-induced (TH)norepinephrine release from superfused rat neocortical slices and synaptosomes were investigated. The adenylate cyclase activator forskolin, the membrane-permeating cyclic AMP analogues 8-bromo-cyclic AMP and dibutyryl cyclic AMP, as well as the phosphodiesterase inhibitors isobutylmethylxanthine and 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrolidone (ZK 62771) enhanced the electrically evoked release of (TH)norepinephrine from superfused rat brain neocortex slices. 8-Bromo-cyclic GMP was without effect on the electrically evoked release. When (TH)norepinephrine release was enhanced by prolonging the electrical pulse duration from 2 msec to 10 msec, the relative inhibitory effect of the CaS channel blocker CdS and the relative facilitatory effect of the K+ channel blocker 4-aminopyridine remained unaffected. In striking contrast, the relative facilitatory effects of forskolin and 8-bromo-cyclic AMP were strongly reduced, whereas the effect of ZK 62771 was almost doubled. When veratrine-induced release of (TH)norepinephrine from cortex synaptosomes was examined, the facilitatory effects of forskolin, 8-bromo-cyclic AMP, and ZK 62771 were even more pronounced than in brain slices. The data strongly support the hypothesis that a presynaptic adenylate cyclase system plays a facilitatory role in the stimulus-secretion coupling process in central noradrenergic nerve terminals.

  10. Effects of Acetazolamide on the Unrinary Excretion of Cyclic AMP and on the Activity of Renal Adenyl Cyclase

    PubMed Central

    Rodriguez, Hector J.; Walls, John; Yates, Jesse; Klahr, Saulo

    1974-01-01

    Acetazolamide, an inhibitor of the enzyme carbonic anhydrase, increased the urinary excretion of cyclic AMP in normal and parathyroidectomized rats. The increase was greater in rats with intact parathyroid glands than in parathyroidectomized rats. This rise in the urinary excretion of cyclic AMP was not due to an increase in urine flow or a change in urine pH. Furosemide caused an increase in urine flow, but did not affect the excretion of cyclic AMP or phosphate. Alkalinization of the urine with bicarbonate did not increase the urinary excretion of phosphate or cyclic AMP. Acetazolamide increased the productionof cyclic AMP by rat renal cortical slices in vitro. This effect was dose-dependent. Acetazolamide also stimulated the activity of renal cortical adenyl cyclase in a dose-dependent manner but had no effect on the activity of cyclic nucleotide phosphodiesterase. The pattern of urinary excretion of cyclic AMP and phosphate after administration of acetazolamide was similar to that observed in rats given parathyroid hormone. It is suggested that acetazolamide stimulates the renal production of cyclic AMP by activating adenyl cyclase and that this may be the mechanism by which this inhibitor of carbonic anhydrase produces phosphaturia. PMID:4357608

  11. Involvement of a membrane-bound class III adenylate cyclase in regulation of anaerobic respiration in Shewanella oneidensis MR-1.

    PubMed

    Charania, M A; Brockman, K L; Zhang, Y; Banerjee, A; Pinchuk, G E; Fredrickson, J K; Beliaev, A S; Saffarini, D A

    2009-07-01

    Unlike other bacteria that use FNR to regulate anaerobic respiration, Shewanella oneidensis MR-1 uses the cyclic AMP receptor protein (CRP) for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases, respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an Escherichia coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, dimethyl sulfoxide (DMSO), or Fe(III), whereas deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III) and, to a lesser extent, with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways, such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagellum biosynthesis, and electron transport were differentially expressed in the cyaC mutant but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration and may contribute to additional signaling pathways independent of CRP.

  12. Involvement of a Membrane-Bound Class III Adenylate Cyclase in Regulation of Anaerobic Respiration in Shewanella oneidensis MR-1

    SciTech Connect

    Charania, M.; Brockman, K. L.; Zhang, Y.; Banerjee, A.; Pinchuk, Grigoriy E.; Fredrickson, Jim K.; Beliaev, Alex S.; Saffarini, Daad

    2009-07-01

    Unlike other bacteria that use FNR to regulate anaerobic respiration, Shewanella oneidensis MR-1 uses the cyclic AMP receptor protein (CRP) for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases, respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an Escherichia coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, dimethyl sulfoxide (DMSO), or Fe(III), whereas deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III) and, to a lesser extent, with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways, such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagellum biosynthesis, and electron transport were differentially expressed in the cyaC mutant but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration and may contribute to additional signaling pathways independent of CRP.

  13. Involvement of a Membrane-Bound Class III Adenylate Cyclase in Regulation of Anaerobic Respiration in Shewanella oneidensis MR-1

    SciTech Connect

    Charania, M.; Brockman, K.; Zhang, Yang; Banerjee, A.; Pinchuk, Grigoriy; Fredrickson, Jim K.; Beliaev, Alex S.; Saffarini, Daad

    2009-07-01

    Unlike other bacteria that use FNR to regulate anaerobic respiration, S. oneidensis MR-1 uses the cAMP receptor protein, CRP, for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an E. coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, DMSO, or Fe(III), whereas the deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III), and to a lesser extent with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and the cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagella biosynthesis, and electron transport, were differentially expressed in the cyaC mutant, but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration, and may contribute to additional signaling pathways independent of CRP.

  14. Dual actions of (-)-stepholidine on the dopamine receptor-mediated adenylate cyclase activity in rat corpus striatum.

    PubMed

    Dong, Z J; Guo, X; Chen, L J; Han, Y F; Jin, G Z

    1997-01-01

    (-)-Stepholidine (SPD) is an antagonist of normosensitive dopamine (DA) receptors, but it exhibits D1 agonistic action on rotational behaviour in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the substantia nigra pars compacta (SNC). In the present study, agonistic and antagonistic effects of SPD on the DA receptor-mediated synaptosomal adenylate cyclase (AC) activity in rat striatum were investigated. After blockade of D2 receptors, SPD augmented AC activity dose-dependently. The EC50 value was 41.1 +/- 8.6 micromol/L. At the concentration of 10 micromol/L, SPD increased cAMP formation from a basal level (50.8 +/- 10.3 pmol/mg protein/min) to 133.7 +/- 31.8 pmol/mg protein/min. The SPD-induced stimulation of AC activity was almost completely reversed by 10 micromol/L Sch23390. These results indicate that SPD possesses an agonistic action on the D1 receptor. Forskolin-stimulated adenylate cyclase (FSAC) activity was used as a model to elucidate the effect of SPD on D2 receptors. The results indicate that DA inhibited FSAC activity dose-dependently, while SPD partially restored FSAC activity. Taken together, these results support the conclusion that SPD has dual actions on DA receptors that mediate AC activity, i.e., an agonistic action on D1 receptors and an antagonistic action on D2 receptors.

  15. Stability, structural and functional properties of a monomeric, calcium–loaded adenylate cyclase toxin, CyaA, from Bordetella pertussis

    PubMed Central

    Cannella, Sara E.; Ntsogo Enguéné, Véronique Yvette; Davi, Marilyne; Malosse, Christian; Sotomayor Pérez, Ana Cristina; Chamot-Rooke, Julia; Vachette, Patrice; Durand, Dominique; Ladant, Daniel; Chenal, Alexandre

    2017-01-01

    Bordetella pertussis, the causative agent of whooping cough, secretes an adenylate cyclase toxin, CyaA, which invades eukaryotic cells and alters their physiology by cAMP overproduction. Calcium is an essential cofactor of CyaA, as it is the case for most members of the Repeat-in-ToXins (RTX) family. We show that the calcium-bound, monomeric form of CyaA, hCyaAm, conserves its permeabilization and haemolytic activities, even in a fully calcium-free environment. In contrast, hCyaAm requires sub-millimolar calcium in solution for cell invasion, indicating that free calcium in solution is involved in the CyaA toxin translocation process. We further report the first in solution structural characterization of hCyaAm, as deduced from SAXS, mass spectrometry and hydrodynamic studies. We show that hCyaAm adopts a compact and stable state that can transiently conserve its conformation even in a fully calcium-free environment. Our results therefore suggest that in hCyaAm, the C-terminal RTX-domain is stabilized in a high-affinity calcium-binding state by the N-terminal domains while, conversely, calcium binding to the C-terminal RTX-domain strongly stabilizes the N-terminal regions. Hence, the different regions of hCyaAm appear tightly connected, leading to stabilization effects between domains. The hysteretic behaviour of CyaA in response to calcium is likely shared by other RTX cytolysins. PMID:28186111

  16. Enhancement of adenylate cyclase activity by phorbol ester: effects on the inhibitory pathway in S49 lymphoma cells

    SciTech Connect

    Bell, J.D.; Brunton, L.L.

    1986-05-01

    12-0-tetradecanoylphorbol-13-acetate (TPA) enhances the apparent V/sub max/ of adenylate cyclase (AC) in S49 lymphoma cells. This effect does not result from an increased rate of activation of the catalytic subunit by the stimulatory GTP binding transducer protein (G/sub s/). In wild type (WT) membranes this enhancement seems to involve a GTP binding protein since TPA enhances forskolin-stimulated AC activity by 30% in the presence of GTP (10 ..mu..M) or Gpp(NH)p (1 ..mu..M) but not in the absence of guanine nucleotide. The authors obtain comparable results in the cyc- variant that lacks the GTP binding subunit of G/sub s/ responsible for stimulating AC, suggesting the importance of a different GTP binding protein. Blockade of the activity of the inhibitory GTP binding protein (G/sub i/) by high concentrations of Mg/sup + +/ (approx.100 mM) or Mn/sup + +/ (approx.1 mM) abolishes the effect of TPA to enhance AC activity in WT membranes. The time course of Gpp(NH)p-mediated inhibition of AC reveals a characteristic lag prior to steady state, indicative of the rate of G/sub i/ activation; TPA increases this lag 3-4 fold. The authors conclude that reduction in the rate of activation of G/sub i/ by guanine nucleotide is one mechanism by which phorbol esters enhance guanine nucleotide-dependent activity of AC, hypothetically via the phosphorylation of G/sub i/ by protein kinase C.

  17. Structure of the RNA 30-Phosphate Cyclase-Adenylate Intermediate Illuminates Nucleotide Specificity and Covalent Nucleotidyl Transfer

    SciTech Connect

    Tanaka, N.; Smith, P; Shuman, S

    2010-01-01

    RNA 3-phosphate cyclase (RtcA) synthesizes RNA 2,3 cyclic phosphate ends via three steps: reaction with ATP to form a covalent RtcA-AMP intermediate; transfer of adenylate to an RNA 3-phosphate to form RNA(3)pp(5)A; and attack of the vicinal O2 on the 3-phosphorus to form a 2,3 cyclic phosphate. Here we report the 1.7 {angstrom} crystal structure of the RtcA-AMP intermediate, which reveals the mechanism of nucleotidyl transfer. Adenylate is linked via a phosphoamide bond to the His309 N{var_epsilon} atom. A network of hydrogen bonds to the ribose O2 and O3 accounts for the stringent ribonucleotide preference. Adenine is sandwiched in a hydrophobic pocket between Tyr284 and Pro131 and the preference for adenine is enforced by Phe135, which packs against the purine C2 edge. Two sulfates bound near the adenylate plausibly mimic the 3-terminal and penultimate phosphates of RNA. The structure illuminates how the four {alpha}2/{beta}4 domains contribute to substrate binding and catalysis.

  18. Regulation of follitropin-sensitive adenylate cyclase by stimulatory and inhibitory forms of the guanine nucleotide regulatory protein in immature rat Sertoli cells

    SciTech Connect

    Johnson, G.P.

    1987-01-01

    Studies have been designed to examine the role of guanine nucleotides in mediating FSH-sensitive adenylate cyclase activity in Sertoli cell plasma membranes. Analysis of ({sup 3}H)GDP binding to plasma membranes suggested a single high affinity site with a K{sub d} = 0.24 uM. Competition studies indicated that GTP{sub {gamma}}S was 7-fold more potent than GDP{sub {beta}}S. Bound GDP could be released by FSH in the presence of GTP{sub {gamma}}S, but not by FSH alone. Adenylate cyclase activity was enhanced 5-fold by FSH in the presence of GTP. Addition of GDP{sub {beta}}S to the activated enzyme (FSH plus GTP) resulted in a time-dependent decay to basal activity within 20 sec. GDP{sub {beta}}S competitively inhibited GTP{sub {gamma}}S-stimulated adenylate cyclase activity with a K{sub i} = 0.18 uM. Adenylate cyclase activity was also demonstrated to be sensitive to the nucleotide bound state. In the presence of FSH, only the GTP{sub {gamma}}S-bound form persisted even if GDP{sub {beta}}S previously occupied all available binding sites. Two membrane proteins, M{sub r} = 43,000 and 48,000, were ADP{centered dot}ribosylated using cholera toxin and labeling was enhanced 2 to 4-fold by GTP{sub {gamma}}S but not by GDP{sub {beta}}S. The M{sub r} = 43,000 and 48,000 proteins represented variant forms of G{sub S}. A single protein of M{sub r} = 40,000 (G{sub i}) was ADP-ribosylated by pertussis toxin in vitro. GTP inhibited forskolin-stimulated adenylate cyclase activity with an IC{sub 50} = 0.1 uM. The adenosine analog, N{sup 6}{centered dot}phenylisopropyl adenosine enhanced GTP inhibition of forskolin-stimulated adenylate cyclase activity by an additional 15%. GTP-dependent inhibition of forskolin-sensitive adenylate cyclase activity was abolished in membranes prepared from Sertoli cells treated in culture with pertussis toxin.

  19. CAP1, an Adenylate Cyclase-Associated Protein Gene, Regulates Bud-Hypha Transitions, Filamentous Growth, and Cyclic AMP Levels and Is Required for Virulence of Candida albicans

    PubMed Central

    Bahn, Yong-Sun; Sundstrom, Paula

    2001-01-01

    In response to a wide variety of environmental stimuli, the opportunistic fungal pathogen Candida albicans exits the budding cycle, producing germ tubes and hyphae concomitant with expression of virulence genes, such as that encoding hyphal wall protein 1 (HWP1). Biochemical studies implicate cyclic AMP (cAMP) increases in promoting bud-hypha transitions, but genetic evidence relating genes that control cAMP levels to bud-hypha transitions has not been reported. Adenylate cyclase-associated proteins (CAPs) of nonpathogenic fungi interact with Ras and adenylate cyclase to increase cAMP levels under specific environmental conditions. To initiate studies on the relationship between cAMP signaling and bud-hypha transitions in C. albicans, we identified, cloned, characterized, and disrupted the C. albicans CAP1 gene. C. albicans strains with inactivated CAP1 budded in conditions that led to germ tube formation in isogenic strains with CAP1. The addition of 10 mM cAMP and dibutyryl cAMP promoted bud-hypha transitions and filamentous growth in the cap1/cap1 mutant in liquid and solid media, respectively, showing clearly that cAMP promotes hypha formation in C. albicans. Increases in cytoplasmic cAMP preceding germ tube emergence in strains having CAP1 were markedly diminished in the budding cap1/cap1 mutant. C. albicans strains with deletions of both alleles of CAP1 were avirulent in a mouse model of systemic candidiasis. The avirulence of a germ tube-deficient cap1/cap1 mutant coupled with the role of Cap1 in regulating cAMP levels shows that the Cap1-mediated cAMP signaling pathway is required for bud-hypha transitions, filamentous growth, and the pathogenesis of candidiasis. PMID:11325951

  20. CAP1, an adenylate cyclase-associated protein gene, regulates bud-hypha transitions, filamentous growth, and cyclic AMP levels and is required for virulence of Candida albicans.

    PubMed

    Bahn, Y S; Sundstrom, P

    2001-05-01

    In response to a wide variety of environmental stimuli, the opportunistic fungal pathogen Candida albicans exits the budding cycle, producing germ tubes and hyphae concomitant with expression of virulence genes, such as that encoding hyphal wall protein 1 (HWP1). Biochemical studies implicate cyclic AMP (cAMP) increases in promoting bud-hypha transitions, but genetic evidence relating genes that control cAMP levels to bud-hypha transitions has not been reported. Adenylate cyclase-associated proteins (CAPs) of nonpathogenic fungi interact with Ras and adenylate cyclase to increase cAMP levels under specific environmental conditions. To initiate studies on the relationship between cAMP signaling and bud-hypha transitions in C. albicans, we identified, cloned, characterized, and disrupted the C. albicans CAP1 gene. C. albicans strains with inactivated CAP1 budded in conditions that led to germ tube formation in isogenic strains with CAP1. The addition of 10 mM cAMP and dibutyryl cAMP promoted bud-hypha transitions and filamentous growth in the cap1/cap1 mutant in liquid and solid media, respectively, showing clearly that cAMP promotes hypha formation in C. albicans. Increases in cytoplasmic cAMP preceding germ tube emergence in strains having CAP1 were markedly diminished in the budding cap1/cap1 mutant. C. albicans strains with deletions of both alleles of CAP1 were avirulent in a mouse model of systemic candidiasis. The avirulence of a germ tube-deficient cap1/cap1 mutant coupled with the role of Cap1 in regulating cAMP levels shows that the Cap1-mediated cAMP signaling pathway is required for bud-hypha transitions, filamentous growth, and the pathogenesis of candidiasis.

  1. Knocking down the expression of adenylate cyclase-associated protein 1 inhibits the proliferation and migration of breast cancer cells.

    PubMed

    Yu, Xia-Fei; Ni, Qi-Chao; Chen, Jin-Peng; Xu, Jun-Fei; Jiang, Ying; Yang, Shu-Yun; Ma, Jing; Gu, Xiao-Ling; Wang, Hua; Wang, Ying-Ying

    2014-04-01

    Adenylate cyclase-associated protein 1 (CAP1) is a conserved protein that was found to be up-regulated in breast cancer and related to the migration of breast cancer. We verified its roles in breast cancer specimens and cell lines. In our results, 71 of 100 specimens of breast cancer showed high levels of CAP1 by immunohistochemistry. Associated with statistical analysis, we saw that CAP1 was related to the grade of breast cancer. In MDA-MB-231, the expression of CAP1 was the highest and by knocking down the expression of CAP1 in MDA-MB-231, its ability for proliferating and migrating apparently decreased and induced changes in morphology, which were related to the arrangement of F-actin. Therefore, CAP1 might be a potential molecular targeted therapy for surgery and immune treatment.

  2. Pituitary adenylate cyclase activating polypeptide in stress-related disorders: data convergence from animal and human studies

    PubMed Central

    May, Victor

    2014-01-01

    The maladaptive expression and function of several stress-associated hormones have been implicated in pathological stress- and anxiety-related disorders. Among these, recent evidence has suggested that pituitary adenylate cyclase activating polypeptide (PACAP) has critical roles in central neurocircuits mediating stress-related emotional behaviors. We describe the PACAPergic systems, the data implicating PACAP in stress biology and how altered PACAP expression and signaling may result in psychopathologies. We include our work implicating PACAP signaling within the bed nucleus of the stria terminalis (BNST) in mediating the consequences of stressor exposure and relatedly, describe more recent studies suggesting that PACAP in the central nucleus of the amygdala (CeA) may impact the emotional aspects of chronic pain states. In aggregate, these results are consistent with data suggesting that PACAP dysregulation is associated with post-traumatic stress disorder (PTSD) in humans. PMID:25636177

  3. Subtyping of Salmonella enterica subspecies I using single nucleotide polymorphisms in adenylate cyclase (cyaA)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methods to rapidly identify serotypes of Salmonella enterica subspecies I are of vital importance for protecting the safety of food. To supplement the serotyping method dkgB-linked intergenic sequence ribotyping (ISR), single nucleotide polymorphisms (SNPs) were characterized within adenylate cyclas...

  4. High expression of adenylate cyclase-associated protein 1 accelerates the proliferation, migration and invasion of neural glioma cells.

    PubMed

    Bao, Zhen; Qiu, Xiaojun; Wang, Donglin; Ban, Na; Fan, Shaochen; Chen, Wenjuan; Sun, Jie; Xing, Weikang; Wang, Yunfeng; Cui, Gang

    2016-04-01

    Adenylate cyclase-associated protein 1 (CAP1), a conserved member of cyclase-associated proteins was reported to be associated with the proliferation, migration or invasion of the tumors of pancreas, breast and liver, and was involved in astrocyte proliferation after acute Traumatic Brain Injury (TBI). In this study, we sought to investigate the character of CAP1 in the pathological process of human glioma by detecting human glioma specimens and cell lines. 43 of 100 specimens showed high expression of CAP1 via immunohistochemistry. With statistics analysis, we found out the expression level of CAP1 was correlated with the WHO grades of human glioma and was great positively related to Ki-67 (p<0.01). In vitro, silencing CAP1 in U251 and U87MG, the glioma cell lines with the relatively higher expression of CAP1, induced the proliferation of the cells significantly retarded, migration and invasion as well. Obviously, our results indicated that CAP1 participated in the molecular pathological process of glioma indeed, and in a certain sense, CAP1 might be a potential and promising molecular target for glioma diagnosis and therapies in the future.

  5. A FRET-Based Method for Probing the Conformational Behavior of an Intrinsically Disordered Repeat Domain from Bordetella pertussis Adenylate Cyclase

    DTIC Science & Technology

    2009-10-22

    2003) Designing repeat proteins: Well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins. J. Mol...A FRET-Based Method for Probing the Conformational Behavior of an Intrinsically Disordered Repeat Domain from Bordetella pertussis Adenylate Cyclase...changes exhibited by intrinsically disordered proteins is necessary as we continue to unravel their myriad biological functions. In repeats in toxin

  6. Human 5-HT7 receptor-induced inactivation of forskolin-stimulated adenylate cyclase by risperidone, 9-OH-risperidone and other "inactivating antagonists".

    PubMed

    Toohey, Nicole; Klein, Michael T; Knight, Jessica; Smith, Carol; Teitler, Milt

    2009-09-01

    We have previously reported on the unusual human 5-hydroxytryptamine(7) (h5-HT(7)) receptor-inactivating properties of risperidone, 9-OH-risperidone, bromocriptine, methiothepin, metergoline, and lisuride. Inactivation was defined as the inability of 10 microM 5-HT to stimulate cAMP accumulation after brief exposure and thorough removal of the drugs from HEK293 cells expressing h5-HT(7) receptors. Herein we report that brief exposure of the h5-HT(7) receptor-expressing cells to inactivating drugs, followed by removal of the drugs, results in potent and efficacious irreversible inhibition of forskolin-stimulated adenylate cyclase activity. Pretreatment, followed by removal of the inactivating drugs inhibited 10 microM forskolin-stimulated adenylate cyclase activity with potencies similar to the drugs' affinities for the h5-HT(7) receptor. The actions of the inactivating drugs were pertussis toxin-insensitive, indicating the lack of G(i) in their mechanism(s) of action. Methiothepin and bromocriptine maximally inhibited 10 microM forskolin-stimulated adenylate cyclase, whereas the other drugs produced partial inhibition, indicating the drugs are inducing slightly different inactive conformations of the h5-HT(7) receptor. Maximal effects of these inactivating drugs occurred within 15 to 30 min of exposure of the cells to the drugs. A G(s)-mediated inhibition of forskolin-stimulated activity has never been reported. The inactivating antagonists seem to induce a stable conformation of the h5-HT(7) receptor, which induces an altered state of G(s), which, in turn, inhibits forskolin-mediated stimulation of adenylate cyclase. These and previous observations indicate that the inactivating antagonists represent a unique class of drugs and may reveal GPCR regulatory mechanisms previously unknown. These drugs may produce innovative approaches to the development of therapeutic drugs.

  7. Irreversible stimulation of adenylate cyclase activity of fat cell membranes of phosphoramidate and phosphonate analogs of GTP.

    PubMed

    Cuatrecasas, P; Bennett, V; Jacobs, S

    1975-01-01

    The ability of 5'-guanylylimidodiphosphate (Gpp(NH)p) to stimulate irreversibly the adenylate cyclease activity of fat cell membranes has been studied by preincubating the membranes with this or related analogs followed by assaying after thoroughly washing the membranes. Activation can occur in a simple Tris-HCl buffer, in the absence of added divalent cations and in the presence of EDTA. Dithiothreitol enhances the apparent degree of activation, perhaps by stabilization. The importance of utilizing optimal conditions for stabilizing enzyme activity, and of measuring the simultaneous changes in the control enzyme, is illustrated. The organomercurial, p-aminophenylmercuric acetate, inhibits profoundly the activity of the native as well as the Gpp(NH)p-stimulated adenylate cyclase, but in both cases subsequent exposure to dithiothreitol restores fully the original enzyme activity. However, the mercurial-inactivated enzyme does not react with Gpp(NP)p, as evidenced by the subsequent restoration of only the control enzyme activity upon exposure to dithiothreitol. Thus, reaction with Gpp(NH)p requires intact sulfhydryl groups, but the activated state is not irreversibly destroyed by the inactivation caused by sulfhydryl blockade. GTP and, less effectively, GDP and ATP inhibit activation by Gpp(NH)p, but interpretations are complicated by the facts that this inhibition is overcome with time and that GTP and ATP can protect potently from spontaneous inactivation. These two nucleotides can be used in the Gpp(NH)p preincubation to stabilize the enzyme. The Gpp(NH)p-activated enzyme cannot be reversed spontaneously during prolonged incubation at 30 degrees C in the absence or presence of GTP, ATP, MgCl2, glycine, dithiothreitol, NaF or EDTA. The strong nucleophile, neutral hydroxylamine, decreases the Gpp(NH)p-activated enzyme activity and no subsequent activation is detected upon re-exposure to the nucleotide.

  8. Corticotropin-releasing factor binding to peripheral tissue and activation of the adenylate cyclase-adenosine 3',5'-monophosphate system

    SciTech Connect

    Dave, J.R.; Eiden, L.E.; Eskay, R.L.

    1985-06-01

    Specific binding sites for rat corticotropin-releasing factor (rCRF) are present in rat adrenal medulla, ventral prostate, spleen, liver, kidney, and testis and bovine chromaffin cells in culture. Maximal binding of (/sup 125/I)rCRF occurred within 25 min at 4 C and was saturable. Scatchard analysis of rCRF binding to rat adrenal membranes and bovine chromaffin cells revealed the existence of two classes of binding sites. One class had a relatively higher apparent affinity and lower number of binding sites, whereas the other class had a relatively lower affinity and higher number of binding sites. CRF induced a dose-related increase in rat adrenal membrane adenylate cyclase activity and cAMP levels in bovine chromaffin cells. Nanomolar concentrations of rCRF maximally stimulated adenylate cyclase activity in rat adrenal membranes and maximally increased cAMP levels in bovine chromaffin cells to 86% and 130% above control values, respectively. The demonstration of specific CRF-binding sites in a variety of peripheral tissues and the finding that activation of specific CRF-binding sites in adrenal tissue stimulates the adenylate cyclase-cAMP system suggest that CRF may have an important regulatory role in various peripheral tissues.

  9. 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.

  10. (/sup 3/H)forskolin- and (/sup 3/H)dihydroalprenolol-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.

    1988-03-01

    The characteristics of the cardiac adenylate cyclase system were studied in rats fed diets containing fish oil (menhaden oil) and other oils. Adenylate cyclase activity generally was higher in cardiac homogenates and membranes of rats fed diet containing 10% menhaden oil than in the other oils. The increase in enzyme activity, especially in forskolin-stimulated activity, was associated with an increase in the concentration of the (/sup 3/H) forskolin-binding sites in cardiac membranes of rats fed menhaden oil. The beta-adrenergic receptor concentration was not significantly altered although the affinity for (/sup 3/H)dihydroalprenolol-binding was lower in membranes of rats fed menhaden oil than those fed the other oils. omega-3 fatty acids from menhaden oil were incorporated into the cardiac membrane phospholipids. The results suggest that the observed increase in myocardial adenylate cyclase activity of rats fed menhaden oil may be due to an increase in the number of the catalytic subunits of the enzyme or due to a greater availability of the forskolin-binding sites.

  11. Defective dopamine-1 receptor adenylate cyclase coupling in the proximal convoluted tubule from the spontaneously hypertensive rat.

    PubMed Central

    Kinoshita, S; Sidhu, A; Felder, R A

    1989-01-01

    The natriuretic effect of DA-1 agonists is less in the spontaneously hypertensive rat (SHR) than its normotensive control, the Wistar-Kyoto rat (WKY). To determine a mechanism of the decreased effect of DA-1 agonists on sodium transport, DA-1 receptors in renal proximal convoluted tubule (PCT) were studied by radioligand binding and by adenylate cyclase (AC) determinations. Specific binding of 125I-SCH 23982 (defined by 10 microM SCH 23390, a DA-1 antagonist) was concentration dependent, saturable, and stereoselective. The dissociation constant, maximum receptor density, and DA-1 antagonist inhibition constant were similar in SHR and WKY. The apparent molecular weight of the DA-1 receptor determined by the photoaffinity D1 probe 125I-MAB was also similar in WKY and SHR. However, DA-1 agonists competed more effectively for specific 125I-SCH 23982 binding sites in WKY than in SHR. Basal as well as forskolin, parathyroid hormone, GTP and Gpp(NH)p-stimulated-AC activities were similar. In contrast DA-1 agonists (fenoldopam, SKF 38393, SND 911C12) stimulated AC activity to a lesser extent in SHR. GTP and Gpp(NH)p enhanced the ability of DA-1 agonists to stimulate AC activity in WKY but not in SHR. These data suggest a defect in the DA-1 receptor-second messenger coupling mechanism in the PCT of the SHR. Images PMID:2574187

  12. Purification and primary structure of pituitary adenylate cyclase activating polypeptide (PACAP) from the brain of an elasmobranch, stingray, Dasyatis akajei.

    PubMed

    Matsuda, K; Yoshida, T; Nagano, Y; Kashimoto, K; Yatohgo, T; Shimomura, H; Shioda, S; Arimura, A; Uchiyama, M

    1998-01-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) was isolated from ovine hypothalami and found to exist as two amidated forms with 38 (PACAP 38) and 27 (PACAP 27) residues. The amino acid sequences of PACAPs isolated from the vertebrates, such as a bird, a frog and teleost fish, appear to be well conserved. In the present study, we attempted to isolate PACAP from the brain of an elasmobranch fish, Dasyatis akajei (stingray), which belongs to the Chondrichthyes (cartilaginous fish), by extraction of the acetone-dried powder with acetic acid, followed by successive high-performance liquid chromatography (HPLC) on a gel-filtration, a cation-exchange and two reverse-phase columns. Purification was monitored by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting analysis using an anti-PACAP 27 serum. The PACAP thus obtained consisted of 44 residues. The amino acid sequence of the comparable portion of its N-terminal 38 residues showed 92%, 89%, 89%, and 82% identity with those of mammalian, chicken, frog and teleost PACAPs with 38 residues, respectively. The extra six C-terminal residues of the stingray resembled those of tetrapod and teleost PACAP precursors which were deduced from the respective cDNAs. These results indicate that PACAP, which has an amino acid sequence showing high similarity with those of tetrapod and teleost PACAPs, is present in the elasmobranch brain.

  13. Role of pituitary adenylate cyclase-activating polypeptide in modulating hypothalamus-pituitary neuroendocrine functions in mouse cell models.

    PubMed

    Kanasaki, H; Oride, A; Kyo, S

    2015-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally identified as a hypothalamic activator of cyclic adenosine monophosphate production in pituitary cells. PACAP and its receptor are expressed not only in the central nervous system, but also in peripheral organs, and function to stimulate pituitary hormone synthesis and secretion as both a hypothalamic-pituitary-releasing factor and an autocrine-paracrine factor within the pituitary. PACAP stimulates the expression of the gonadotrophin α, luteinising hormone (LH) β and follicle-stimulating hormone (FSH) β subunits, as well as the gonadotrophin-releasing hormone (GnRH) receptor and its own PACAP type I receptor (PAC1R) in gonadotrophin-secreting pituitary cells. In turn, GnRH, which is known to be a crucial component of gonadotrophin secretion, stimulates the expression of PACAP and PAC1R in gonadotrophs. In addition, PAC1R and PACAP modulate the functions of GnRH-producing neurones in the hypothalamus. This review summarises the current understanding of the possible roles of PACAP and PAC1R in modulating hypothalamus and pituitary neuroendocrine cells in the mouse models.

  14. Rapid, semi-automated, and inexpensive radioimmunoassay of cAMP: application in GPCR-mediated adenylate cyclase assays.

    PubMed

    Brown, Justin T; Kant, Andrew; Mailman, Richard B

    2009-03-15

    Cyclic AMP (cAMP) is an important signal transduction second messenger that is commonly used as a functional mirror on the actions of G protein-coupled receptors that can activate or inhibit adenylate cyclases. A radioimmunoassay for cAMP with femtomole sensitivity was first reported by Steiner more than 30 years ago, and there have been several subsequent modifications that have improved this assay in various ways. Here we describe additional improvement to existing methods that markedly improve speed and reduce cost without sacrificing sensitivity, and is also adaptable to analysis of cGMP. The primary antibody is coupled directly to magnetic beads that are then separated from unbound marker using filtration on microplates. This eliminates the need for a secondary antibody, and markedly increases throughput. In addition, we report a simple, reproducible, and inexpensive method to make the radiomarker used for this assay. Although still requiring the use of radioactivity, the resulting method retains a high degree of accuracy and precision, and is suitable for low-cost high throughput screening. Use of aspects of this method can also improve throughput in other radioimmunoassays.

  15. 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.

  16. Pharmacological characterization of the dopamine-sensitive adenylate cyclase in cockroach brain: evidence for a distinct dopamine receptor

    SciTech Connect

    Orr, G.L.; Gole, J.W.D.; Notman, H.J.; Downer, R.G.H.

    1987-12-21

    Dopamine increases cyclic AMP production in crude membrane preparations of cockroach brain with plateaus in cyclic AMP production occurring between 1-10 ..mu..M and 10 mM. Maximal production of cyclic AMP is 2.25 fold greater than that of control values. Octopamine also increases cyclic AMP production with a Ka of 1.4 ..mu..M and maximal production 3.5 fold greater than that of control. 5-Hydroxytryptamine does not increase cyclic AMP production. The effects of octopamine and dopamine are fully additive. The vertebrate dopamine agonists ADTN and epinine stimulate the dopamine-sensitive adenylate cyclase (AC) with Ka values of 4.5 and 0.6 ..mu..M respectively and with maximal effectiveness 1.7 fold greater than that of control. The selective D/sub 2/-dopamine agonist LY-171555 stimulates cyclic AMP production to a similar extent with a Ka of 50 ..mu..M. Other dopamine agonists have no stimulatory effects. With the exception of mianserin, /sup 3/H-piflutixol is displaced from brain membranes by dopamine antagonists with an order of potency similar to that observed for the inhibition of dopamine-sensitive AC. The results indicate that the octopamine- and dopamine-sensitive AC in cockroach brain can be distinguished pharmacologically and the dopamine receptors coupled to AC have pharmacological characteristics distinct from vertebrate D/sup 1/- and D/sup 2/-dopamine receptors. 33 references, 3 figures, 2 tables.

  17. Oxidative Stress Tolerance, Adenylate Cyclase, and Autophagy Are Key Players in the Chronological Life Span of Saccharomyces cerevisiae during Winemaking

    PubMed Central

    Orozco, Helena; Matallana, Emilia

    2012-01-01

    Most grape juice fermentation takes place when yeast cells are in a nondividing state called the stationary phase. Under such circumstances, we aimed to identify the genetic determinants controlling longevity, known as the chronological life span. We identified commercial strains with both short (EC1118) and long (CSM) life spans in laboratory growth medium and compared them under diverse conditions. Strain CSM shows better tolerance to stresses, including oxidative stress, in the stationary phase. This is reflected during winemaking, when this strain has an increased maximum life span. Compared to EC1118, CSM overexpresses a mitochondrial rhodanese gene-like gene, RDL2, whose deletion leads to increased reactive oxygen species production at the end of fermentation and a correlative loss of viability at this point. EC1118 shows faster growth and higher expression of glycolytic genes, and this is related to greater PKA activity due to the upregulation of the adenylate cyclase gene. This phenotype has been linked to the presence of a δ element in its promoter, whose removal increases the life span. Finally, EC1118 exhibits a higher level of protein degradation by autophagy, which might help achieve fast growth at the expense of cellular structures and may be relevant for long-term survival under winemaking conditions. PMID:22327582

  18. Stress tolerance of the Saccharomyces cerevisiae adenylate cyclase fil1 (CYR1) mutant depends on Hsp26.

    PubMed

    Vianna, Cristina R; Ferreira, Mariana C; Silva, Carol L C; Tanghe, An; Neves, Maria J; Thevelein, Johan M; Rosa, Carlos A; Van Dijck, Patrick

    2010-01-01

    Fermentation-induced loss of stress resistance in yeast is an important phenotype from an industrial point of view. It hampers optimal use of frozen dough applications as well as high gravity brewing fermentations because these applications require stress-tolerant yeast strains during active fermentation. Different mutants (e.g. fil1, an adenylate cyclase mutant CYR1(lys1682)) that are affected in this loss of stress resistance have been isolated, but so far the identification of the target genes important for the increased tolerance has failed. Previously we have shown that neither trehalose nor Hsp104 nor STRE-controlled genes are involved in the higher stress tolerance of the fil1 mutant. The contribution of other putative downstream factors of the PKA pathway was investigated and here we show that the small heat-shock protein Hsp26 is required for the high heat stress tolerance of the fil1 mutant, both in stationary phase cells as well as during active fermentation.

  19. The vasorelaxant effect of pituitary adenylate cyclase activating polypeptide and vasoactive intestinal polypeptide in isolated rat basilar arteries is partially mediated by activation of nitrergic neurons.

    PubMed

    Seebeck, Jörg; Löwe, Marcus; Kruse, Marie Luise; Schmidt, Wolfgang E; Mehdorn, H Maximilian; Ziegler, Albrecht; Hempelmann, Ralf G

    2002-07-15

    The structurally related neuropeptides pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are recognised by two G protein-coupled receptors, termed VPAC(1)-R and VPAC(2)-R, with equal affinity. PACAP and VIP have previously been shown to relax cerebral arteries in an endothelium-independent manner. The aim of the present study was to test if intramural neurons are involved in the mediation of PACAP/VIP-induced vasodilatory responses. Therefore, the vascular tone of isolated rat basilar arteries was measured by means of a myograph. The vasorelaxing effect of PACAP was assessed in arteries precontracted by serotonin in the absence or presence of different test compounds known to selectively inhibit certain signaling proteins. The vasorelaxant effect of PACAP could be significantly reduced by the inhibitor of neuronal N-type calcium channels omega-conotoxin GVIA (omega-CgTx), as well as by 3-bromo-7-nitroindazole (3Br-7-Ni), an inhibitor of the neuronal nitric oxide-synthase (nNOS). The localization of N-type calcium channels and VPAC-Rs within the rat basilar artery was investigated by confocal laser scanning microscopy using omega-CgTx- and VIP-analogs labelled with fluorescent dyes. These findings suggest that activation of intramural neurons may represent an important effector mechanism for mediation of the vasorelaxant PACAP-response.

  20. The relationship between the occupation of the D-1 dopamine receptor by [3H]piflutixol and the activity of dopamine-sensitive adenylate cyclase in rat striatal membranes.

    PubMed

    Fleminger, S

    1991-07-05

    The relationship between occupation of the D-1 dopamine receptor by [3H]piflutixol and inhibition of dopamine-sensitive adenylate cyclase has been studied. Experiments were performed in parallel; after the initial incubation to enable binding of [3H]piflutixol, half the tubes were assayed for [3H]piflutixol binding and the other half assayed for adenylate cyclase activity. The assay conditions for the two halves of the experiments were identical. (+/-)Sulpiride (3 x 10(-5)M) was present in all tubes to mask drug binding to the D-2 receptor. The inhibition of dopamine- (10(-3) and 10(-5)M) sensitive adenylate cyclase with increasing concentrations of [3H]piflutixol in the incubation mixture was compared to the saturation of specific [3H]piflutixol binding with those same concentrations of [3H]piflutixol. There was a linear relationship between receptor occupation by [3H]piflutixol and inhibition of dopamine sensitive adenylate cyclase. In a second experiment dopamine was present during the initial incubation with [3H]piflutixol. This resulted in a displacement of specific [3H]piflutixol binding and, as a consequence, a reduction of [3H]piflutixol's inhibition of dopamine-sensitive adenylate cyclase. In the absence of GTP in the initial incubation dopamine produced a greater reduction of [3H]piflutixol's inhibition of dopamine adenylate cyclase than displacement of specific [3H]piflutixol binding. In the presence of GTP in the initial incubation both displacement curves were shifted to the right, i.e. dopamine was less potent. However, under these conditions dopamine produced less inhibition of [3H]piflutixol's inhibition of dopamine adenylate cyclase than displacement of specific [3H]piflutixol binding. These results are interpreted as resulting from changes in D-1high and D-1low ratios as a result of incubation in the presence or absence of GTP.

  1. In vivo control of gluconeogenesis in wild-type Neurospora crassa and in the adenylate cyclase-deficient cr-1 (crisp) mutant.

    PubMed Central

    Neves, M J; Terenzi, H F

    1989-01-01

    The rate of cycloheximide-resistant incorporation of carbon from [14C]alanine and [14C]acetate into polysaccharidic material was used to study gluconeogenic activity in wild-type Neurospora crassa and in the adenylate cyclase-deficient cr-1 (crisp-1) mutant. The wild-type efficiently utilized alanine and acetate as gluconeogenic substrates, whereas the mutant used acetate efficiently but was unable to use alanine. Cycloheximide-resistant 14C-incorporating activity was sensitive to carbon catabolite effects (repression and inactivation) in the two strains, which suggested that cyclic AMP metabolism was not involved in these regulatory responses. In the wild type, gluconeogenesis was induced by incubation of the cells in the absence of a carbon source. In contrast, cr-1 required supplementation with acetate. This finding suggested that induction of gluconeogenesis in N. crassa could be mediated by metabolites formed in carbon-starved cells. The cr-1 mutant seemed to be deficient in this process and to depend on an exogenous effector to induce gluconeogenesis. Incubation of cr-1 with cyclic AMP partially overcame the acetate requirement for induction of gluconeogenesis. PMID:2522093

  2. Toxicity of Cry1A toxins from Bacillus thuringiensis to CF1 cells does not involve activation of adenylate cyclase/PKA signaling pathway.

    PubMed

    Portugal, Leivi; Muñóz-Garay, Carlos; Martínez de Castro, Diana L; Soberón, Mario; Bravo, Alejandra

    2017-01-01

    Bacillus thuringiensis (Bt) bacteria produce Cry toxins that are able to kill insect pests. Different models explaining the mode of action of these toxins have been proposed. The pore formation model proposes that the toxin creates pores in the membrane of the larval midgut cells after interaction with different receptors such as cadherin, aminopeptidase N and alkaline phosphatase and that this pore formation activity is responsible for the toxicity of these proteins. The alternative model proposes that interaction with cadherin receptor triggers an intracellular cascade response involving protein G, adenylate cyclase (AC) and protein kinase A (PKA). In addition, it was shown that Cry toxins induce a defense response in the larvae involving the activation of mitogen-activated kinases such as MAPK p38 in different insect orders. Here we analyzed the mechanism of action of Cry1Ab and Cry1Ac toxins and a collection of mutants from these toxins in the insect cell line CF1 from Choristoneura fumiferana, that is naturally sensitive to these toxins. Our results show that both toxins induced permeability of K(+) ions into the cells. The initial response after intoxication with Cry1Ab and Cry1Ac toxins involves the activation of a defense response that involves the phosphorylation of MAPK p38. Analysis of activation of PKA and AC activities indicated that the signal transduction involving PKA, AC and cAMP was not activated during Cry1Ab or Cry1Ac intoxication. In contrast we show that Cry1Ab and Cry1Ac activate apoptosis. These data indicate that Cry toxins can induce an apoptotic death response not related with AC/PKA activation. Since Cry1Ab and Cry1Ac toxins affected K(+) ion permeability into the cells, and that mutant toxins affected in pore formation are not toxic to CF1, we propose that pore formation activity of the toxins is responsible of triggering cell death response in CF1cells.

  3. Protective Effects of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Against Oxidative Stress in Zebrafish Hair Cells.

    PubMed

    Kasica, Natalia; Podlasz, Piotr; Sundvik, Maria; Tamas, Andrea; Reglodi, Dora; Kaleczyc, Jerzy

    2016-11-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide, with known antiapoptotic functions. Our previous in vitro study has demonstrated the ameliorative role of PACAP-38 in chicken hair cells under oxidative stress conditions, but its effects on living hair cells is now yet known. Therefore, the aim of the present study was to investigate in vivo the protective role of PACAP-38 in hair cells found in zebrafish (Danio rerio) sense organs-neuromasts. To induce oxidative stress the 5-day postfertilization (dpf) zebrafish larvae were exposed to 1.5 mM H2O2 for 15 min or 1 h. This resulted in an increase in caspase-3 and p-38 MAPK level in the hair cells as well as in an impairment of the larvae basic behavior. To investigate the ameliorative role of PACAP-38, the larvae were incubated with a mixture of 1.5 mM H2O2 and 100 nM PACAP-38 following 1 h preincubation with 100 nM PACAP-38 only. PACAP-38 abilities to prevent hair cells from apoptosis were investigated. Whole-mount immunohistochemistry and confocal microscopy analyses revealed that PACAP-38 treatment decreased the cleaved caspase-3 level in the hair cells, but had no influence on p-38 MAPK. The analyses of basic locomotor activity supported the protective role of PACAP-38 by demonstrating the improvement of the fish behavior after PACAP-38 treatment. In summary, our in vivo findings demonstrate that PACAP-38 protects zebrafish hair cells from oxidative stress by attenuating oxidative stress-induced apoptosis.

  4. Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) Signalling Exerts Chondrogenesis Promoting and Protecting Effects: Implication of Calcineurin as a Downstream Target

    PubMed Central

    Juhász, Tamás; Matta, Csaba; Katona, Éva; Somogyi, Csilla; Takács, Roland; Gergely, Pál; Csernoch, László; Panyi, Gyorgy; Tóth, Gábor; Reglődi, Dóra; Tamás, Andrea; Zákány, Róza

    2014-01-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is an important neurotrophic factor influencing differentiation of neuronal elements and exerting protecting role during traumatic injuries or inflammatory processes of the central nervous system. Although increasing evidence is available on its presence and protecting function in various peripheral tissues, little is known about the role of PACAP in formation of skeletal components. To this end, we aimed to map elements of PACAP signalling in developing cartilage under physiological conditions and during oxidative stress. mRNAs of PACAP and its receptors (PAC1,VPAC1, VPAC2) were detectable during differentiation of chicken limb bud-derived chondrogenic cells in micromass cell cultures. Expression of PAC1 protein showed a peak on days of final commitment of chondrogenic cells. Administration of either the PAC1 receptor agonist PACAP 1-38, or PACAP 6-38 that is generally used as a PAC1 antagonist, augmented cartilage formation, stimulated cell proliferation and enhanced PAC1 and Sox9 protein expression. Both variants of PACAP elevated the protein expression and activity of the Ca-calmodulin dependent Ser/Thr protein phosphatase calcineurin. Application of PACAPs failed to rescue cartilage formation when the activity of calcineurin was pharmacologically inhibited with cyclosporine A. Moreover, exogenous PACAPs prevented diminishing of cartilage formation and decrease of calcineurin activity during oxidative stress. As an unexpected phenomenon, PACAP 6-38 elicited similar effects to those of PACAP 1-38, although to a different extent. On the basis of the above results, we propose calcineurin as a downstream target of PACAP signalling in differentiating chondrocytes either in normal or pathophysiological conditions. Our observations imply the therapeutical perspective that PACAP can be applied as a natural agent that may have protecting effect during joint inflammation and/or may promote cartilage regeneration

  5. Presence of pituitary adenylate cyclase-activating polypeptide (PACAP) in the plasma and milk of ruminant animals.

    PubMed

    Czegledi, Levente; Tamas, Andrea; Borzsei, Rita; Bagoly, Terez; Kiss, Peter; Horvath, Gabriella; Brubel, Reka; Nemeth, Jozsef; Szalontai, Balint; Szabadfi, Krisztina; Javor, Andras; Reglodi, Dora; Helyes, Zsuzsanna

    2011-05-15

    Milk contains a variety of proteins and peptides that possess biological activity. Growth factors, such as growth hormone, insulin-like, epidermal and nerve growth factors are important milk components which may regulate growth and differentiation in various neonatal tissues and also those of the mammary gland itself. We have recently shown that pituitary adenylate cyclase-activating polypeptide (PACAP), an important neuropeptide with neurotrophic actions, is present in the human milk in much higher concentration than in the plasma of lactating women. Investigation of growth factors in the milk of domestic animals is of utmost importance for their nutritional values and agricultural significance. Therefore, the aim of the present study was to determine the presence and concentration of PACAP in the plasma and milk of three ruminant animal species. Furthermore, the presence of PACAP and its specific PAC1 receptor were investigated in the mammary glands. Radioimmunoassay measurements revealed that PACAP was present in the plasma and the milk of the sheep, goat and the cow in a similar concentration to that measured previously in humans. PACAP38-like immunoreactivity (PACAP38-LI) was 5-20-fold higher in the milk than in the plasma samples of the respective animals, a similar serum/milk ratio was found in all the three species. The levels did not show significant changes within the examined 3-month-period of lactation after delivery. Similar PACAP38-LI was measured in the homogenates of the sheep mammary gland samples taken 7 and 30 days after delivery. PAC1 receptor expression was detected in these udder biopsies by fluorescent immunohistochemistry suggesting that this peptide might have an effect on the mammary glands themselves. These data show that PACAP is present in the milk of various ruminant domestic animal species at high concentrations, the physiological implications of which awaits further investigation.

  6. Pituitary adenylate cyclase-activating polypeptide prevents the effects of ceramides on migration, neurite outgrowth, and cytoskeleton remodeling.

    PubMed

    Falluel-Morel, Anthony; Vaudry, David; Aubert, Nicolas; Galas, Ludovic; Benard, Magalie; Basille, Magali; Fontaine, Marc; Fournier, Alain; Vaudry, Hubert; Gonzalez, Bruno J

    2005-02-15

    During neuronal migration, cells that do not reach their normal destination or fail to establish proper connections are eliminated through an apoptotic process. Recent studies have shown that the proinflammatory cytokine tumor necrosis factor alpha (and its second messengers ceramides) and the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) play a pivotal role in the histogenesis of the cerebellar cortex. However, the effects of ceramides and PACAP on migration of cerebellar granule cells have never been investigated. Time-lapse videomicroscopy recording showed that C2-ceramide, a cell-permeable ceramide analog, and PACAP induced opposite effects on cell motility and neurite outgrowth. C2-ceramide markedly stimulated cell movements during the first hours of treatment and inhibited neuritogenesis, whereas PACAP reduced cell migration and promoted neurite outgrowth. These actions of C2-ceramide on cell motility and neurite outgrowth were accompanied by a disorganization of the actin filament network, depolarization of tubulin, and alteration of the microtubule-associated protein Tau. In contrast, PACAP strengthened the polarization of actin at the emergence cone, increased Tau phosphorylation, and abolished C2-ceramide-evoked alterations of the cytoskeletal architecture. The caspase-inhibitor Z-VAD-FMK, like PACAP, suppressed the "dance of the death" provoked by C2-ceramide. Finally, Z-VAD-FMK and the PP2A inhibitor okadaic acid both prevented the impairment of Tau phosphorylation induced by C2-ceramide. Taken together, these data indicate that the reverse actions of C2-ceramide and PACAP on cerebellar granule cell motility and neurite outgrowth are attributable to their opposite effects on actin distribution, tubulin polymerization, and Tau phosphorylation.

  7. Cloning, tissue distribution and effects of fasting on pituitary adenylate cyclase-activating polypeptide in largemouth bass

    NASA Astrophysics Data System (ADS)

    Li, Shengjie; Han, Linqiang; Bai, Junjie; Ma, Dongmei; Quan, Yingchun; Fan, Jiajia; Jiang, Peng; Yu, Lingyun

    2015-03-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) has a wide range of biological functions. We cloned the full-length cDNAs encoding PACAP and PACAP-related peptide (PRP) from the brain of largemouth bass ( Micropterus salmoides) and used real-time quantitative PCR to detect PRP-PACAP mRNA expression. The PRP-PACAP cDNA has two variants expressed via alternative splicing: a long form, which encodes both PRP and PACAP, and a short form, which encodes only PACAP. Sequence analysis results are consistent with a higher conservation of PACAP than PRP peptide sequences. The expression of PACAP-long and PACAP-short transcripts was highest in the forebrain, followed by the medulla, midbrain, pituitary, stomach, cerebellum, intestine, and kidney; however, these transcripts were either absent or were weakly expressed in the muscle, spleen, gill, heart, fatty tissue, and liver. The level of PACAP-short transcript expression was significantly higher than expression of the long transcript in the forebrain, cerebella, pituitary and intestine, but lower than that of the long transcript in the stomach. PACAP-long and PACAP-short transcripts were first detected at the blastula stage of embryogenesis, and the level of expression increased markedly between the muscular contraction stage and 3 d post hatch (dph). The expression of PACAP-long and PACAP-short transcripts decreased significantly in the brain following 4 d fasting compared with the control diet group. The down-regulation effect was enhanced as fasting continued. Conversely, expression levels increased significantly after 3 d of re-feeding. Our results suggest that PRP-PACAP acts as an important factor in appetite regulation in largemouth bass.

  8. Overexpression of adenylate cyclase-associated protein 1 is associated with metastasis of lung cancer.

    PubMed

    Tan, Min; Song, Xiaolian; Zhang, Guoliang; Peng, Aimei; Li, Xuan; Li, Ming; Liu, Yang; Wang, Changhui

    2013-10-01

    Lung cancer ranks first in both prevalence and mortality rates among all types of cancer. Metastasis is the main cause of treatment failure. Biomarkers are critical to early diagnosis and prediction and monitoring of progressive lesions. Several biomarkers have been identified for lung cancer but none have been routinely used clinically. The present study assessed the diagnostic and prognostic value of cyclase-associated protein 1 (CAP1) for lung cancer. CAP1 mRNA abundance and protein content were determined by real-time PCR and western blot analysis and/or immunostaining in biopsy specimens (24 neoplastic and 6 non-neoplastic) freshly collected at surgical lung resection, in 82 pathologically banked lung cancer specimens and in cultured non-invasive (95-C) and invasive (95-D) lung cancer cells. Multivariate regression analysis was performed to correlate immunoreactive CAP1 signal with cancer type and stage. In vitro cell migration was performed to determine the effect of RNA interference-mediated CAP1 gene silencing on invasiveness of 95-D cells. These analyses collectively demonstrated that: i) both CAP1 mRNA abundance and protein content were significantly higher in neoplastic compared to non-neoplastic specimens and in metastatic compared to non-metastatic specimens but not different between adenocarcinoma and squamous cell carcinoma; ii) immunoreactive CAP1 signal was significantly stronger in metastatic specimens and 95-D cells compared to non-metastatic specimens and 95-C cells; and iii) RNA interference-mediated CAP1 gene silencing adequately attenuated the invasive capacity of 95-D cells in vitro. These findings suggest that overexpression of CAP1 in lung cancer cells, particularly at the metastatic stage, may have significant clinical implications as a diagnostic/prognostic factor for lung cancer.

  9. Centrally acting hypotensive agents with affinity for 5-HT1A binding sites inhibit forskolin-stimulated adenylate cyclase activity in calf hippocampus.

    PubMed Central

    Schoeffter, P.; Hoyer, D.

    1988-01-01

    1. A number of centrally acting hypotensive agents and other ligands with high affinity for 5-hydroxytryptamine1A (5-HT1A) recognition sites have been tested on forskolin-stimulated adenylate cyclase activity in calf hippocampus, a functional model for 5-HT1A-receptors. 2. Concentration-dependent inhibition of forskolin-stimulated adenylate cyclase activity was elicited by the reference 5-HT1-receptor agonists (mean EC50 value, nM): 5-HT (22), 5-carboxamidotryptamine (5-CT, 3.2), 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT, 8.6), N,N-dipropyl-5-carboxamidotryptamine (DP-5-CT, 2.3), 1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)-piperazine (PAPP or LY 165163, 20), 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H indole (RU 24969, 20), buspirone (65) and ipsapirone (56). Emax amounted to 18-20% inhibition for all but the latter two agonists (14%). 3. The following hypotensive agents with high affinity for 5-HT1A sites were potent agonists in this system (mean EC50 value, nM): flesinoxan (24), indorenate (99), erythro-1-(1-[2-(1,4-benzodioxan-2-yl)-2-hydroxyethyl]-4-piperidyl )- 2-benzimidazolinone (R 28935, 2.5), urapidil (390) and 5-methyl-urapidil (3.5). The first two agents were full agonists, whereas the latter three acted as partial agonists with 60-80% efficacy. 4. Metergoline and methysergide behaved as full agonists and cyanopindolol as a partial agonist with low efficacy. Spiroxatrine and 2-(2,6-dimethoxyphenoxyethyl)aminomethyl- 1,4-benzodioxane (WB 4101) which bind to 5-HT1A sites with nanomolar affinity, were agonists and inhibited potently forskolin-stimulated adenylate cyclase in calf hippocampus, showing mean EC50 values of 23 and 15 nM, respectively. Spiroxatrine and WB 4101 yielded 90% and 50% efficacy, respectively. 5. Spiperone and methiothepin (each 1 microM) caused rightward shifts of the concentration-effect curve to 8-OH-DPAT, without loss of the maximal effect, as did the partial agonist cyanopindolol (0.1 microM) and the

  10. 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 separate 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.

  11. Bi-directional effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on fear-related behavior and c-Fos expression after fear conditioning in rats

    PubMed Central

    Meloni, Edward G.; Venkataraman, Archana; Donahue, Rachel J.; Carlezon, William A.

    2015-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is implicated in stress regulation and learning and memory. PACAP has neuromodulatory actions on brain structures within the limbic system that could contribute to its acute and persistent effects in animal models of stress and anxiety-like behavior. Here, male Sprague-Dawley rats were implanted with intracerebroventricular (ICV) cannula for infusion of PACAP-38 (0.5, 1, or 1.5 ug) or vehicle followed 30 min later by fear conditioning. Freezing was measured early (1, 4, and 7days) or following a delay (7, 10, and 13 days)after conditioning. PACAP (1.5 μg) produced a bi-phasic response in freezing behavior across test days: relative to controls, PACAP-treated rats showed a reduction in freezing when tested 1 or 7 Days after fear conditioning that evolved into a significant elevation in freezing by the third test session in the early, but not delayed, group. Corticosterone (CORT) levels were significantly elevated in PACAP-treated rats following fear conditioning, but not at the time of testing (Day 1). Brain c-Fos expression revealed PACAP-dependent alterations within, as well as outside of, areas typically implicated in fear conditioning. Our findings raise the possibility that PACAP disrupts fear memory consolidation by altering synaptic plasticity within neurocircuits normally responsible for encoding fear-related cues, producing a type of dissociation or peritraumatic amnesia often seen in people early after exposure to a traumatic event. However, fear memories are retained such that repeated testing and memory reactivation (e.g. re-experiencing) causes the freezing response to emerge and persist at elevated levels. PACAP systems may represent an axis on which stress and exposure to trauma converge to promote maladaptive behavioral responses characteristic of psychiatric illnesses such as post-traumatic stress disorder (PTSD). PMID:26590791

  12. ASP-56, a new actin sequestering protein from pig platelets with homology to CAP, an adenylate cyclase-associated protein from yeast.

    PubMed

    Gieselmann, R; Mann, K

    1992-02-24

    A new 56 kDa actin-binding protein (ASP-56) was isolated from pig platelet lysate. In falling ball viscosimetry it caused a reduction in viscosity that could be attributed to a decrease in the concentration of polymeric actin. Fluorescence measurements with NBD-labelled actin showed reduction of polymeric actin, too. These results could be explained by sequestering of actin in a non-polymerizable 1:1 ASP-56/actin complex. Sequencing of about 20 tryptic peptides of ASP-56 and comparison with known sequences revealed about 60% homology to the adenylate cyclase-associated protein (CAP) from yeast.

  13. Absorption and fluorescence characteristics of photo-activated adenylate cyclase nano-clusters from the amoeboflagellate Naegleria gruberi NEG-M strain

    NASA Astrophysics Data System (ADS)

    Penzkofer, A.; Stierl, M.; Hegemann, P.; Kateriya, S.

    2012-01-01

    The spectroscopic characteristics of BLUF (BLUF = sensor of blue light using flavin) domain containing soluble adenylate cyclase (nPAC = Naegleria photo-activated cyclase) samples from the amoeboflagellate Naegleria gruberi NEG-M strain is studied at room temperature. The absorption and fluorescence spectroscopic development in the dark was investigated over two weeks. Attenuation coefficient spectra, fluorescence quantum distributions, fluorescence quantum yields, and fluorescence excitation distributions were measured. Thawing of frozen nPAC samples gave solutions with varying protein nano-cluster size and varying flavin, tyrosine, tryptophan, and protein color-center emission. Protein color-center emission was observed in the wavelength range of 360-900 nm with narrow emission bands of small Stokes shift and broad emission bands of large Stokes shift. The emission spectra evolved in time with protein nano-cluster aging.

  14. Part I: Pituitary adenylate cyclase-activating polypeptide-38 induced migraine-like attacks in patients with and without familial aggregation of migraine.

    PubMed

    Guo, Song; Vollesen, Anne Luise Haulund; Hansen, Rikke Dyhr; Esserlind, Ann-Louise; Amin, Faisal Mohammed; Christensen, Anne Francke; Olesen, Jes; Ashina, Messoud

    2017-02-01

    Background Intravenous infusion of adenylate cyclase-activating polypeptide-38 (PACAP38) provokes migraine-like attacks in 65-70% of migraine sufferers. Whether aggregation of migraine in first-degree relatives contributes to this discrepancy in PACAP38-induced response is unknown. We hypothesized that genetic enrichment plays a role in triggering of migraine and that migraine without aura patients with a high family load ( ≥ 2 first-degree relatives with migraine) would report more migraine-like attacks after intravenous infusion of human PACAP38. Methods In this study, we allocated 32 previously genotyped migraine without aura patients to receive intravenous infusion of 10 pmol/kg/min PACAP38 and recorded migraine-like attacks including headache characteristics and associated symptoms. Information of familial aggregation was obtained by telephone interview of first-degree relatives using a validated semi-structured questionnaire. Results PACAP38 infusion induced a migraine-like attack in 75% (nine out of 12) of patients with high family load compared to 70% (14 out of 20) with low family load ( P = 0.761). In an explorative investigation, we found that the migraine response after PACAP38 was not associated with the risk allele of rs2274316 ( MEF2D), which confers increased risk of migraine without aura and may regulate PACAP38 expression. Conclusion Migraine response to PACAP38 infusion in migraine without aura patients is not associated with high family load or the risk allele of rs2274316 ( MEF2D).

  15. Parabrachial nucleus (PBn) pituitary adenylate cyclase activating polypeptide (PACAP) signaling in the amygdala: implication for the sensory and behavioral effects of pain

    PubMed Central

    Missig, Galen A.; Roman, Carolyn W.; Vizzard, Margaret A.; Braas, Karen M.; May, Victor

    2015-01-01

    The intricate relationships that associate pain, stress responses and emotional behavior have been well established. Acute stressful situations can decrease nociceptive sensations and conversely, chronic pain can enhance other pain experiences and heighten the emotional and behavioral consequences of stress. Accordingly, chronic pain is comorbid with a number of behavioral disorders including depression, anxiety abnormalities and associated stress-related disorders including post traumatic stress disorder (PTSD). The central nucleus of the amygdala (CeA) represents a convergence of pathways for pain, stress and emotion, and we have identified pituitary adenylate cyclase activating polypeptide (PACAP) immunoreactivity in fiber elements in the lateral capsular division of the CeA (CeLC). The PACAP staining patterns colocalized in part with those for calcitonin gene related peptide (CGRP); anterograde fiber tracing and excitotoxic lesion studies demonstrated that the CeLC PACAP/CGRP immunoreactivities represented sensory fiber projections from the lateral parabrachial nucleus (LPBn) along the spino-parabrachioamygdaloid tract. The same PBn PACAP/CGRP fiber system also projected to the BNST. As in the BNST, CeA PACAP signaling increased anxiety-like behaviors accompanied by weight loss and decreased feeding. But in addition to heightened anxiety-like responses, CeA PACAP signaling also altered nociception as reflected by decreased latency and threshold responses in thermal and mechanical sensitivity tests, respectively. From PACAP expression in major pain pathways, the current observations are novel and suggest that CeA PACAP nociceptive signaling and resulting neuroplasticity via the spino-parabrachioamygdaloid tract may represent mechanisms that associate chronic pain with sensory hypersensitivity, fear memory consolidation and severe behavioral disorders. PMID:24998751

  16. Characteristics of muscarinic receptors that selectively couple to inhibition of adenylate cyclase or stimulation of phospholipase C on NG108-15 and 1321N1 cells

    SciTech Connect

    Liang, M.

    1988-01-01

    The purpose of this dissertation was to establish whether different muscarinic receptor proteins selectively couple to different second messenger response system. Although both second messenger response systems are fully functional in both cell lines, activation of muscarinic cholinergic receptors only results in inhibition of adenylate cyclase in NG108-15 neuroblastoma {times} glioma cells and stimulation of phosphoinositide hydrolysis in 1321N1 human astrocytoma cells. Muscarinic receptors on both cell types were covalently labeled with ({sup 3}H)Propylbenzilylcholine mustard (({sup 3}H)PBCM) and the mobilities of the ({sup 3}H)PBCM-labelled species of both cells were compared by SDS-PAGE. 1321N1 and NG108-15 cells each primarily expressed a single ({sup 3}H)PBCM-labelled species with an apparent size of approximately 92,000 and 66,000 Da, respectively. ({sup 3}H)PBCM labelling was completely inhibited by 1 {mu}M atropine or by down-regulation of muscarinic receptors by an overnight incubation with carbachol. The apparent size of the ({sup 3}H)PBCM-labelled species of both cell lines was not altered by treatment with a series of protease inhibitors or by treatment with dithiothreitol and iodoacetamide. Another approach for determining differences in the muscarinic receptors of 2 cells lines was to study agonist-induced alteration of muscarinic receptor number. Exposure of both cell types to agonists resulted in rapid loss of muscarinic receptors from cell surface without change of total cellular muscarinic receptors followed by subsequently loss of receptors from cells. Muscarinic receptors on both cell lines were regulated by agonist with similar properties.

  17. Pituitary Adenylate Cyclase-Activating Polypeptide Induces the Voltage-Independent Activation of Inward Membrane Currents and Elevation of Intracellular Calcium in HIT-T15 Insulinoma Cells*

    PubMed Central

    LEECH, COLIN A.; HOLZ, GEORGE G.; HABENER, JOEL F.

    2010-01-01

    The secretion of insulin by pancreatic β-cells is controlled by synergistic interactions of glucose and hormones of the glucagon-related peptide family, of which pituitary adenylate cyclase-activating polypeptide (PACAP) is a member. Here we show by simultaneous recording of intracellular calcium ion ([Ca2+]i) and membrane potential that both PACAP-27 and PACAP-38 depolarize HIT-T15 cells and raise [Ca2+]i. PACAP stimulation can result in membrane depolarization by two distinct mechanisms: 1) PACAP reduces the membrane conductance and increases membrane excitability; and 2) PACAP activates a pronounced inward current that is predominantly a Na+ current, blockable by La3+, and which exhibits a reversal potential of about −28 mV. Activation of this current does not require membrane depolarization, because the response is observed when cells are held under voltage clamp at −70 mV. This current may result from the cAMP-dependent activation of nonspecific cation channels because the current is also observed in response to forskolin or membrane-permeant analogs of cAMP. We also suggest that PACAP raises [Ca2+]i and stimulates insulin secretion by three distinct mechanisms: 1) depolarization activates Ca2+ influx through L-type voltage-dependent calcium channels, 2) mobilization of intracellular Ca2+ stores, and 3) entry of Ca2+ via voltage-independent Ca2+ channels. These effects of PACAP may play an important role in a neuro-entero-endocrine loop regulating insulin secretion from pancreatic β-cells during the transition period from fasting to feeding. PMID:7895663

  18. Gustatory Habituation in "Drosophila" Relies on "Rutabaga" (Adenylate Cyclase)-Dependent Plasticity of GABAergic Inhibitory Neurons

    ERIC Educational Resources Information Center

    Paranjpe, Pushkar; Rodrigues, Veronica; VijayRaghavan, K.; Ramaswami, Mani

    2012-01-01

    In some situations, animals seem to ignore stimuli which in other contexts elicit a robust response. This attenuation in behavior, which enables animals to ignore a familiar, unreinforced stimulus, is called habituation. Despite the ubiquity of this phenomenon, it is generally poorly understood in terms of the underlying neural circuitry. Hungry…

  19. Effects of pituitary adenylate cyclase activating polypeptide-27 (PACAP) and vasoactive intestinal polypeptide (VIP) on chloride in HT29 cells studied by X-ray microanalysis.

    PubMed

    Zhang, W; Roomans, G M

    1999-01-01

    The colon cancer cell line HT29 is a useful model to study intestinal chloride secretion. These cells have both cAMP-activated and calcium-activated chloride channels. Changes in elemental content of the cells after stimulation with agonists were determined by X-ray microanalysis in the scanning or scanning transmission electron microscope. Exposure of HT29 cells to pituitary adenylate cyclase activating polypeptide-27 (PACAP) caused a transient decrease in the cellular Cl and K concentrations, indicating (net) efflux of chloride. The effect of PACAP is inhibited by somatostatin, which is known to inhibit cAMP-activated as well as calcium-activated chloride secretion and by U-73122, an inhibitor of phospholipase C. Alloxan, an inhibitor of adenylate cyclase, did not significantly affect the PACAP-induced loss of chloride. The calcium-chelating agent EGTA inhibited the PACAP-induced loss of chloride, indicating the need for extracellular calcium ions. Also vasointestinal polypeptide (VIP) caused a decrease of the cellular chloride concentration in HT29 cells. VIP-induced loss of chloride could be inhibited by pre-treating the cells with somatostatin or UK14,304, an alpha-2 adrenergic agonist that has been shown previously to inhibit purinergically activated chloride efflux. Our results indicate that there is cross-talk between the cAMP- and the calcium-activated pathways for chloride secretion in HT29 cells.

  20. [The effect of hypoxia on the urokinase and adenylate cyclase systems in the culture of endothelial cells of the human umbilical vein].

    PubMed

    Kapustin, A N; Tishchenko, E P; Torosian, N A; Panina, O B; Tsokolaeva, Z I; Ratner, E I; Savel'eva, G M; Parfenova, E V

    2005-06-01

    Hypoxia induces angiogenesis in ischemized tissues by means of pro-angiogenic factor expression. The key role in the growth processes and blood vessel functioning belongs to the matrix metalloproteinases, plasminogen, and its activator systems. Effect of hypoxia on expression of the urokinase activating agent plasminogen and its receptor in endothelium was studied in human umbilical vein endothelial cell model. Incubation of the endothelial cells under the conditions of hypoxia proved to reduce both urokinase formation in these cells and its secreting into the culture medium. The hypoxia-induced reduction of urokinase contents was accompanied by enhancement of expression of the urokinase receptor. The hypoxia also entailed reduction of the adenylate cyclase activity and cAMP contents in the endothelial cells. The data obtained suggest that reduction of the adenylate cyclase activity and cAMP contents under the conditions of hypoxia provide basis for suppression of the urokinase expression by the endothelial cells and, consequently, inhibition of blood vessel formation in the ischemized tissue.

  1. 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 phospholipase 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.

  2. Negatively charged residues of the segment linking the enzyme and cytolysin moieties restrict the membrane-permeabilizing capacity of adenylate cyclase toxin

    PubMed Central

    Masin, Jiri; Osickova, Adriana; Sukova, Anna; Fiser, Radovan; Halada, Petr; Bumba, Ladislav; Linhartova, Irena; Osicka, Radim; Sebo, Peter

    2016-01-01

    The whooping cough agent, Bordetella pertussis, secretes an adenylate cyclase toxin-hemolysin (CyaA) that plays a crucial role in host respiratory tract colonization. CyaA targets CR3-expressing cells and disrupts their bactericidal functions by delivering into their cytosol an adenylate cyclase enzyme that converts intracellular ATP to cAMP. In parallel, the hydrophobic domain of CyaA forms cation-selective pores that permeabilize cell membrane. The invasive AC and pore-forming domains of CyaA are linked by a segment that is unique in the RTX cytolysin family. We used mass spectrometry and circular dichroism to show that the linker segment forms α-helical structures that penetrate into lipid bilayer. Replacement of the positively charged arginine residues, proposed to be involved in target membrane destabilization by the linker segment, reduced the capacity of the toxin to translocate the AC domain across cell membrane. Substitutions of negatively charged residues then revealed that two clusters of negative charges within the linker segment control the size and the propensity of CyaA pore formation, thereby restricting the cell-permeabilizing capacity of CyaA. The ‘AC to Hly-linking segment’ thus appears to account for the smaller size and modest cell-permeabilizing capacity of CyaA pores, as compared to typical RTX hemolysins. PMID:27581058

  3. Identification of a prostacyclin receptor coupled to the adenylate cyclase system via a stimulatory GTP-binding protein in mouse mastocytoma P-815 cells

    SciTech Connect

    Hashimoto, H.; Negishi, M.; Ichikawa, A. )

    1990-11-01

    A stable analogue of prostacyclin, iloprost, specifically bound to 30,000 x g pellet (the membrane fraction) prepared from mouse mastocytoma P-815 cells. The binding was dependent on time, temperature and pH, and absolutely required a divalent cation. The equilibrium dissociation constant and the maximal concentration of the binding site as determined by Scatchard plot analysis were 10.4 nM and 1.12 pmol/mg of protein, respectively. The Hill coefficient was 1.0, indicating a single entity of binding site and no cooperativity. The binding site was highly specific for iloprost among PGs tested (iloprost much greater than PGE1 greater than carbacyclin greater than PGE2). In contrast, the membrane fraction had the binding site specific for PGE2 and PGE1, which was distinct from the prostacyclin receptor. The dissociation of bound (3H)iloprost from the membrane fraction was specifically enhanced by guanine nucleotides. Furthermore, iloprost dose-dependently enhanced the activity of adenylate cyclase in a GTP-dependent manner. These results indicate that a specific prostacyclin receptor is coupled to the adenylate cyclase system via a stimulatory GTP-binding protein in mastocytoma cells.

  4. Bisamidate Prodrugs of 2-Substituted 9-[2-(Phosphonomethoxy)ethyl]adenine (PMEA, adefovir) as Selective Inhibitors of Adenylate Cyclase Toxin from Bordetella pertussis.

    PubMed

    Česnek, Michal; Jansa, Petr; Šmídková, Markéta; Mertlíková-Kaiserová, Helena; Dračínský, Martin; Brust, Tarsis F; Pávek, Petr; Trejtnar, František; Watts, Val J; Janeba, Zlatko

    2015-08-01

    Novel small-molecule agents to treat Bordetella pertussis infections are highly desirable, as pertussis (whooping cough) remains a serious health threat worldwide. In this study, a series of 2-substituted derivatives of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA, adefovir), in their isopropyl ester bis(L-phenylalanine) prodrug form, were designed and synthesized as potent inhibitors of adenylate cyclase toxin (ACT) isolated from B. pertussis. The series consists of PMEA analogues bearing either a linear or branched aliphatic chain or a heteroatom at the C2 position of the purine moiety. Compounds with a small C2 substituent showed high potency against ACT without cytotoxic effects as well as good selectivity over human adenylate cyclase isoforms AC1, AC2, and AC5. The most potent ACT inhibitor was found to be the bisamidate prodrug of the 2-fluoro PMEA derivative (IC50 =0.145 μM). Although the bisamidate prodrugs reported herein exhibit overall lower activity than the bis(pivaloyloxymethyl) prodrug (adefovir dipivoxil), their toxicity and plasma stability profiles are superior. Furthermore, the bisamidate prodrug was shown to be more stable in plasma than in macrophage homogenate, indicating that the free phosphonate can be effectively distributed to target tissues, such as the lungs. Thus, ACT inhibitors based on acyclic nucleoside phosphonates may represent a new strategy to treat whooping cough.

  5. Properties of rat erythrocyte membrane cytoskeletal structures produced by digitonin extraction: digitonin-insoluble beta-adrenergic receptor, adenylate cyclase, and cholera toxin substrate.

    PubMed

    LeVine, H; Sahyoun, N E; Cuatrecasas, P

    1982-01-01

    Rat erythrocyte plasma membranes have been extracted exhaustively with digitonin at low temperature, and the residual, detergent-extracted membrane cytoskeletal material is compared to that prepared with Triton X-100 with respect to protein, glycoprotein, phospholipid, and cholesterol content. Digitonin, a weaker detergent than Triton X-100, solubilizes only 26% of the phospholipids and none of the cholesterol. SDS-polyacrylamide gel electrophoresis reveals that differences between the proteins extracted by the two detergents are primarily quantitative. In terms of functional preservation, digitonin retains in the cytoskeleton 28% of the beta-adrenergic receptor binding activity (with the balance accounted for in the supernatant), greater than 90% of the adenylate cyclase and greater than 90% of the 45,000 mol wt polypeptide cholera toxin substrate. The cytoskeletal-associated beat-adrenergic receptor retains binding properties for antagonist and agonist which are identical to those of the native membrane receptor. The digitonin-extracted cytoskeleton containing the beta-adrenergic receptor may provide a useful vehicle for the reconstitution of a hormone-sensitive adenylate cyclase.

  6. Pituitary Adenylate Cyclase-Activating Peptide in the Central Amygdala Causes Anorexia and Body Weight Loss via the Melanocortin and the TrkB Systems.

    PubMed

    Iemolo, Attilio; Ferragud, Antonio; Cottone, Pietro; Sabino, Valentina

    2015-07-01

    Growing evidence suggests that the pituitary adenylate cyclase-activating polypeptide (PACAP)/PAC1 receptor system represents one of the main regulators of the behavioral, endocrine, and autonomic responses to stress. Although induction of anorexia is a well-documented effect of PACAP, the central sites underlying this phenomenon are poorly understood. The present studies addressed this question by examining the neuroanatomical, behavioral, and pharmacological mechanisms mediating the anorexia produced by PACAP in the central nucleus of the amygdala (CeA), a limbic structure implicated in the emotional components of ingestive behavior. Male rats were microinfused with PACAP (0-1 μg per rat) into the CeA and home-cage food intake, body weight change, microstructural analysis of food intake, and locomotor activity were assessed. Intra-CeA (but not intra-basolateral amygdala) PACAP dose-dependently induced anorexia and body weight loss without affecting locomotor activity. PACAP-treated rats ate smaller meals of normal duration, revealing that PACAP slowed feeding within meals by decreasing the regularity and maintenance of feeding from pellet-to-pellet; postprandial satiety was unaffected. Intra-CeA PACAP-induced anorexia was blocked by coinfusion of either the melanocortin receptor 3/4 antagonist SHU 9119 or the tyrosine kinase B (TrKB) inhibitor k-252a, but not the CRF receptor antagonist D-Phe-CRF(12-41). These results indicate that the CeA is one of the brain areas through which the PACAP system promotes anorexia and that PACAP preferentially lessens the maintenance of feeding in rats, effects opposite to those of palatable food. We also demonstrate that PACAP in the CeA exerts its anorectic effects via local melanocortin and the TrKB systems, and independently from CRF.

  7. Regulation of the beta-adrenergic receptor-adenylate cyclase complex of 3T3-L1 fibroblasts by sodium butyrate

    SciTech Connect

    Stadel, J.M.; Poksay, K.S.; Nakada, M.T.; Crooke, S.T.

    1986-05-01

    Mouse 3T3-L1 fibroblasts contain beta-adrenergic receptors (BAR), predominantly of the B/sub 1/ subtype. Incubation of these cells with 2-10 mM sodium butyrate (SB) for 24-48 hr results in a switch in the BAR subtype from B/sub 1/ to B/sub 2/ and promotes a 1.5 to 2.5 fold increase in total BAR number. Other short chain acids were not as effective as SB in promoting changes in BAR. BAR were assayed in membranes prepared from the 3T3-L1 cells using the radiolabeled antagonist (/sup 125/I)-cyanopindolol and the B/sub 2/ selective antagonist ICI 118.551. BAR subtype switch was confirmed functionally by measuring cellular cAMP accumulation in response to agonists. The structure and amount of the alpha subunits of the guanine nucleotide regulatory proteins N/sub s/ and N/sub i/ were determined by ADP-ribosylation using /sup 32/P-NAD and either cholera toxin or pertussis toxin for labeling of the respective subunits. Preincubation of cells with 5 mM SB for 48 hr resulted in a 2-3 fold increase in the labeling of the alpha subunits of both N/sub s/ and N/sub i/. A protein of M/sub r/ = 44,000 showed enhanced labeling by cholera toxin following SB treatment of the cells. These data indicate SB concomitantly regulates expression of BAR subtype and components of the adenylate cyclase in 3T3-L1 cells.

  8. Characterization of the adenosine receptor in cultured embryonic chick atrial myocytes: Coupling to modulation of contractility and adenylate cyclase activity and identification by direct radioligand binding

    SciTech Connect

    Liang, B.T.

    1989-06-01

    Adenosine receptors in a spontaneously contracting atrial myocyte culture from 14-day chick embryos were characterized by radioligand binding studies and by examining the involvement of G-protein in coupling these receptors to a high-affinity state and to the adenylate cyclase and the myocyte contractility. Binding of the antagonist radioligand (3H)-8-cyclopentyl-1,3-diproylxanthine ((3H)CPX) was rapid, reversible and saturable and was to a homogeneous population of sites with a Kd value of 2.1 +/- 0.2 nM and an apparent maximum binding of 26.2 +/- 3 fmol/mg of protein (n = 10, +/- S.E.). Guanyl-5-yl-(beta, gamma-imido)diphosphate had no effect on either the Kd or the maximum binding and CPX reversed the N6-R-phenyl-2-propyladenosine-induced inhibition of adenylate cyclase activity and contractility, indicating that (3H) CPX is an antagonist radioligand. Competition curves for (3H) CPX binding by a series of reference adenosine agonists were consistent with labeling of an A1 adenosine receptor and were better fit by a two-site model than by a one-site model. ADP-ribosylation of the G-protein by the endogenous NAD+ in the presence of pertussis toxin shifted the competition curves from bi to monophasic with Ki values similar to those of the KL observed in the absence of prior pertussis intoxication. The adenosine agonists were capable of inhibiting both the adenylate cyclase activity and myocyte contractility in either the absence or the presence of isoproterenol. The A1 adenosine receptor-selective antagonist CPX reversed these agonist effects. The order of ability of the reference adenosine receptor agonists in causing these inhibitory effects was similar to the order of potency of the same agonists in inhibiting the specific (3H)CPX binding (N6-R-phenyl-2-propyladenosine greater than N6-S-phenyl-2-propyladenosine or N-ethyladenosine-5'-uronic acid).

  9. Pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP-receptor type 1 expression in rat and human placenta.

    PubMed

    Scaldaferri, M L; Modesti, A; Palumbo, C; Ulisse, S; Fabbri, A; Piccione, E; Frajese, G; Moretti, C

    2000-03-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP), the new hypophysiotropic factor member of the vasoactive intestinal peptide (VIP)/secretin/glucagon/GHRH family of neuropeptides, exerts its biological action by interacting with both PACAP-selective type I receptors (PAC1) and type II receptors (VPAC1), which bind both PACAP and VIP. The placenta is a site of production of hypophysiotropic factors that participate in the control of local hormone production, as well as the respective hypothalamic-pituitary neurohormones. In the present study, we show the expression of PACAP gene and irPACAP distribution within rat and human placental tissues, by means of RT-PCR and immunohystochemical experiments. In both rat and human placenta, we evaluated the expression of PAC1 gene by Northern hybridization analysis performed with a 32P-labeled 706 nt complementary DNA probe, derived from the full-length coding region of the rPAC1 complementary DNA. The results of these experiments demonstrate the presence, in both human and rat placenta, of a 7.5-kb transcript similar in size to those detected in the ovary, brain, and hypothalamus. Alternative splicing of two exons occurs in human and rat PAC1 gene generating splice variants with variable tissue-specific expression. To ascertain which of the splice variants were expressed in placental tissue we performed RT-nested PCR using primers flanking the insertion sequence termed hip/hop cassette in rat or SV1/SV2 box in human gene. Electrophoretic analysis of the PCR products showed a different pattern of expression of messenger RNA splicing variants in human and rat placenta. In particular, the rat placenta expresses the short PAC1 receptor (PAC1short), the rPAC1-hip or hop (which are indistinguishable with the primers used), and the rPAC1-hip-hop, whereas the human placenta expresses only the PAC1SV1 (or SV2) variant, structurally homologous to the rat PAC1 hip (or hop). Sequence analysis of the human PCR-amplified PAC1

  10. Multiple nickel-sensitive targets elicit cardiac arrhythmia in isolated mouse hearts after pituitary adenylate cyclase-activating polypeptide-mediated chronotropy.

    PubMed

    Tevoufouet, Etienne E; Nembo, Erastus N; Distler, Fabian; Neumaier, Felix; Hescheler, Jürgen; Nguemo, Filomain; Schneider, Toni

    2017-03-01

    The pituitary adenylate cyclase-activating polypeptide (PACAP)-27 modulates various biological processes, from the cellular level to function specification. However, the cardiac actions of this neuropeptide are still under intense studies. Using control (+|+) and mice lacking (-|-) either R-type (Cav2.3) or T-type (Cav3.2) Ca(2+) channels, we investigated the effects of PACAP-27 on cardiac activity of spontaneously beating isolated perfused hearts. Superfusion of PACAP-27 (20nM) caused a significant increase of baseline heart frequency in Cav2.3(+|+) (156.9±10.8 to 239.4±23.4 bpm; p<0.01) and Cav2.3(-|-) (190.3±26.4 to 270.5±25.8 bpm; p<0.05) hearts. For Cav3.2, the heart rate was significantly increased in Cav3.2(-|-) (133.1±8.5 bpm to 204.6±27.9 bpm; p<0.05) compared to Cav3.2(+|+) hearts (185.7±11.2 bpm to 209.3±22.7 bpm). While the P wave duration and QTc interval were significantly increased in Cav2.3(+|+) and Cav2.3(-|-) hearts following PACAP-27 superfusion, there was no effect in Cav3.2(+|+) and Cav3.2(-|-) hearts. The positive chronotropic effects observed in the four study groups, as well as the effect on P wave duration and QTc interval were abolished in the presence of Ni(2+) (50μM) and PACAP-27 (20nM) in hearts from Cav2.3(+|+) and Cav2.3(-|-) mice. In addition to suppressing PACAP's response, Ni(2+) also induced conduction disturbances in investigated hearts. In conclusion, the most Ni(2+)-sensitive Ca(2+) channels (R- and T-type) may modulate the PACAP signaling cascade during cardiac excitation in isolated mouse hearts, albeit to a lesser extent than other Ni(2+)-sensitive targets.

  11. In vivo and in vitro models of demyelinating disease: activation of the adenylate cyclase system influences JHM virus expression in explanted rat oligodendrocytes.

    PubMed Central

    Beushausen, S; Narindrasorasak, S; Sanwal, B D; Dales, S

    1987-01-01

    The specificity of JHM virus (JHMV) tropism for rat oligodendrocytes, as one of the primary host cells in the central nervous system, is maintained after explanation (S. Beushausen and S. Dales, Virology 141:89-101, 1985). The temporal correlation between onset of resistance to JHMV infection in vivo, completion of myelination, and maturation of the central nervous system can be simulated in vitro by inducers of oligodendrocyte differentiation (Beushausen and Dales, Virology, 1985). Stimulation of differentiation through the elevation of intracellular cyclic AMP (cAMP) levels suggests a possible connection between activation of the adenylate cyclase system and coronavirus expression. Chromatographic analysis of cAMP-dependent protein kinase activity in cytosol extracts prepared from astrocytes or oligodendrocytes revealed that both glial cell types were deficient in protein kinase I, indicating that expression of coronavirus in differentiated cells was not contingent upon the presence of protein kinase I. However, treatment with N6,2'-O-dibutyryladenosine-3',5'-cyclic monophosphate (dbcAMP) resulted in a severalfold enhancement of the free regulatory subunit (RI) in oligodendrocytes but not in astrocytes. The RII subunit in both neural cell types was relatively unaffected. Rapid increase in RI due to dbcAMP treatment was correlated with inhibition of JHMV expression. Other differentiation inducers, including 8-Br cAMP and forskolin which, by contrast, caused a decrease in detectable RI, also blocked JHMV expression. This apparent anomaly can be attributed to an increased turnover of RI due to destabilization of the molecule which occurs upon site-specific binding of the cyclic nucleotides. On the basis of these observations, we conclude that the state of oligodendrocyte differentiation manifested with the modulation of RI regulates JHMV expression. The differentiation process did not affect either virus adsorption or sequestration but appeared to inhibit the

  12. Saturated high-fat diet-induced obesity increases adenylate cyclase of myocardial β-adrenergic system and does not compromise cardiac function.

    PubMed

    Vileigas, Danielle F; de Deus, Adriana F; da Silva, Danielle C T; de Tomasi, Loreta C; de Campos, Dijon H S; Adorni, Caroline S; de Oliveira, Scarlet M; Sant'Ana, Paula G; Okoshi, Katashi; Padovani, Carlos R; Cicogna, Antonio C

    2016-09-01

    Obesity is a worldwide pandemic associated with high incidence of cardiovascular disease. The mechanisms by which the obesity leads cardiac dysfunction are not fully elucidated and few studies have evaluated the relationship between obesity and proteins involved in myocardial β-adrenergic (βA) system. The purpose of this study was to evaluate the cardiac function and βA pathway components in myocardium of obese rats. Male Wistar rats were distributed into two groups: control (n = 17; standard diet) and obese (n = 17; saturated high-fat diet) fed for 33 weeks. Nutritional profile and comorbidities were assessed. Cardiac structure and function was evaluated by macroscopic postmortem, echocardiographic and isolated papillary muscle analyzes. Myocardial protein expression of β1- and β2-adrenergic receptors, Gαs protein, adenylate cyclase (AC) and protein kinase A (PKA) was performed by Western blot. Cardiac cyclic adenosine monophosphate (cAMP) levels and PKA activity were assessed by ELISA Obese rats showed increased adiposity index (P < 0.001) and several comorbidities as hypertension, glucose intolerance, insulin resistance, and dyslipidemia compared with control rats. Echocardiographic assessment revealed increased left atrium diameter (C: 4.98 ± 0.38 vs. Ob: 5.47 ± 0.53, P = 0.024) and posterior wall shortening velocity (C: 37.1 ± 3.6 vs. Ob: 41.8 ± 3.8, P = 0.007) in obese group. Papillary muscle evaluation indicated that baseline data and myocardial responsiveness to isoproterenol stimulation were similar between the groups. Protein expression of myocardial AC was higher in obese group than in the control (C: 1.00 ± 0.21 vs. Ob: 1.25 ± 0.10, P = 0.025), whereas the other components were unchanged. These results suggest that saturated high-fat diet-induced obesity was not effective in triggering cardiac dysfunction and impair the beta-adrenergic signaling.

  13. 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.

  14. 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

  15. Molecular cloning and mRNA distribution of pituitary adenylate cyclase-activating polypeptide (PACAP)/PACAP-related peptide in the lungfish.

    PubMed

    Lee, L T O; Tam, J K V; Chan, D W; Chow, B K C

    2009-04-01

    In this article, we report the isolation of a full-length cDNA clone encoding pituitary adenylate cyclase-activating polypeptide (PACAP)/PACAP-related peptide (PRP) from lungfish Protopterus dolloi. When comparing the deduced amino acid sequences, the lungfish PACAP was found to be highly conserved with other vertebrates; however, the PRP shares only lower levels of sequence identity with known PRP sequences. Consistently in phylogenetic analysis, the lungfish PRP, similar to sturgeon PRP, fails to cluster with other PRPs. In addition to the full-length clone, another cDNA encoding a short precursor that lacks the first 32 amino acids of the PRP was also isolated. Interestingly, similar isoforms were also identified in several nonmammalian vertebrates, and it was suggested that exon skipping of PRP/PACAP transcripts was a mechanism that regulated the expression ratio of PACAP to PRP in nonmammalian vertebrates. By real-time PCR, both long and short PRP/PACAP transcripts were found almost exclusively in the brain, and the short isoform is the more abundant transcript (3.7 times more), indicating that PACAP is the major product produced in lungfish brain. The expression patterns of lungfish and previously studied frog PRP/PACAP suggest that the PRP/PACAP gene in the tetrapod lineage may first express in the central nervous system; in the process of evolution, the functions of these peptides diversified and were later found in other tissues.

  16. Pituitary adenylate cyclase activating polypeptide (PACAP) dilates cerebellar arteries through activation of large-conductance Ca(2+)-activated (BK) and ATP-sensitive (K ATP) K (+) channels.

    PubMed

    Koide, Masayo; Syed, Arsalan U; Braas, Karen M; May, Victor; Wellman, George C

    2014-11-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a potent vasodilator of numerous vascular beds, including cerebral arteries. Although PACAP-induced cerebral artery dilation is suggested to be cyclic AMP (cAMP)-dependent, the downstream intracellular signaling pathways are still not fully understood. In this study, we examined the role of smooth muscle K(+) channels and hypothesized that PACAP-mediated increases in cAMP levels and protein kinase A (PKA) activity result in the coordinate activation of ATP-sensitive K(+) (KATP) and large-conductance Ca(2+)-activated K(+) (BK) channels for cerebral artery dilation. Using patch-clamp electrophysiology, we observed that PACAP enhanced whole-cell KATP channel activity and transient BK channel currents in freshly isolated rat cerebellar artery myocytes. The increased frequency of transient BK currents following PACAP treatment is indicative of increased intracellular Ca(2+) release events termed Ca(2+) sparks. Consistent with the electrophysiology data, the PACAP-induced vasodilations of cannulated cerebellar artery preparations were attenuated by approximately 50 % in the presence of glibenclamide (a KATP channel blocker) or paxilline (a BK channel blocker). Further, in the presence of both blockers, PACAP failed to cause vasodilation. In conclusion, our results indicate that PACAP causes cerebellar artery dilation through two mechanisms: (1) KATP channel activation and (2) enhanced BK channel activity, likely through increased Ca(2+) spark frequency.

  17. Pituitary Adenylate Cyclase-Activating Polypeptide Regulates Brain-Derived Neurotrophic Factor Exon IV Expression through the VPAC1 Receptor in the Amphibian Melanotrope Cell

    PubMed Central

    Kidane, Adhanet H.; Roubos, Eric W.; Jenks, Bruce G.

    2008-01-01

    In mammals, pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors PAC1-R, VPAC1-R, and VPAC2-R play a role in various physiological processes, including proopiomelanocortin (POMC) and brain-derived neurotrophic factor (BDNF) gene expression. We have previously found that PACAP stimulates POMC gene expression, POMC biosynthesis, and α-MSH secretion in the melanotrope cell of the amphibian Xenopus laevis. This cell hormonally controls the process of skin color adaptation to background illumination. Here, we have tested the hypothesis that PACAP is involved in the regulation of Xenopus melanotrope cell activity during background adaptation and that part of this regulation is through the control of the expression of autocrine acting BDNF. Using quantitative RT-PCR, we have identified the Xenopus PACAP receptor, VPAC1-R, and show that this receptor in the melanotrope cell is under strong control of the background light condition, whereas expression of PAC1-R was absent from these cells. Moreover, we reveal by quantitative immunocytochemistry that the neural pituitary lobe of white-background adapted frogs possesses a much higher PACAP content than the neural lobe of black-background adapted frogs, providing evidence that PACAP produced in the hypothalamic magnocellular nucleus plays an important role in regulating the activity of Xenopus melanotrope cells during background adaptation. Finally, an in vitro study demonstrates that PACAP stimulates the expression of BDNF transcript IV. PMID:18450956

  18. Atomoxetine reverses locomotor hyperactivity, impaired novel object recognition, and prepulse inhibition impairment in mice lacking pituitary adenylate cyclase-activating polypeptide.

    PubMed

    Shibasaki, Y; Hayata-Takano, A; Hazama, K; Nakazawa, T; Shintani, N; Kasai, A; Nagayasu, K; Hashimoto, R; Tanida, M; Katayama, T; Matsuzaki, S; Yamada, K; Taniike, M; Onaka, Y; Ago, Y; Waschek, J A; Köves, K; Reglődi, D; Tamas, A; Matsuda, T; Baba, A; Hashimoto, H

    2015-06-25

    Attention-deficit/hyperactivity disorder (ADHD) is a complex neurobehavioral disorder that is characterized by attention difficulties, impulsivity, and hyperactivity. A non-stimulant drug, atomoxetine (ATX), which is a selective noradrenaline reuptake inhibitor, is widely used for ADHD because it exhibits fewer adverse effects compared to conventional psychostimulants. However, little is known about the therapeutic mechanisms of ATX. ATX treatment significantly alleviated hyperactivity of pituitary adenylate cyclase-activating polypeptide (PACAP)-deficient (PACAP(-/-)) mice with C57BL/6J and 129S6/SvEvTac hybrid background. ATX also improved impaired novel object recognition memory and prepulse inhibition in PACAP(-/-) mice with CD1 background. The ATX-induced increases in extracellular noradrenaline and dopamine levels were significantly higher in the prefrontal cortex of PACAP(-/-) mice compared to wild-type mice with C57BL/6J and 129S6/SvEvTac hybrid background. These results suggest that ATX treatment-induced increases in central monoamine metabolism may be involved in the rescue of ADHD-related abnormalities in PACAP(-/-) mice. Our current study suggests that PACAP(-/-) mice are an ideal rodent model with predictive validity for the study of ADHD etiology and drug development. Additionally, the potential effects of differences in genetic background of PACAP(-/-) mice on behaviors are discussed.

  19. Reduced expression of brain-derived neurotrophic factor in mice deficient for pituitary adenylate cyclase activating polypeptide type-I-receptor.

    PubMed

    Zink, Mathias; Otto, Christiane; Zörner, Björn; Zacher, Christiane; Schütz, Günther; Henn, Fritz A; Gass, Peter

    2004-04-22

    In vitro pituitary adenylate cyclase activating polypeptide (PACAP) induces the expression of brain-derived neurotrophic factor (BDNF) via its specific receptor PAC1. Since BDNF has been implicated in learning paradigms and mice lacking functional PAC1 have deficits in hippocampus-dependent associative learning, we investigated whether PAC1 mutants show alterations in hippocampal expression of BDNF and its receptor TrkB. Semi-quantitative in situ-hybridization using exon-specific BDNF-probes revealed significantly reduced expression of the exon-III and exon-V-specific transcripts within the hippocampal CA3 region in PAC1-deficient mice. A similar trend was observed for the exon-I-specific transcript. The expression of the exon-III-specific transcript was also reduced within the dentate gyrus, while Trk B-expression did not differ between genotypes. Our data demonstrate that even in vivo PAC1-mediated signaling seems to play a pivotal role for the transcriptional regulation of BDNF.

  20. Effect of the pituitary adenylate cyclase-activating polypeptide on the autophagic activation observed in in vitro and in vivo models of Parkinson's disease.

    PubMed

    Lamine-Ajili, Asma; Fahmy, Ahmed M; Létourneau, Myriam; Chatenet, David; Labonté, Patrick; Vaudry, David; Fournier, Alain

    2016-04-01

    Parkinson's disease (PD) is a neurodegenerative disorder that leads to destruction of the midbrain dopaminergic (DA) neurons. This phenomenon is related to apoptosis and its activation can be blocked by the pituitary adenylate cyclase-activating polypeptide (PACAP). Growing evidence indicates that autophagy, a self-degradation activity that cleans up the cell, is induced during the course of neurodegenerative diseases. However, the role of autophagy in the pathogenesis of neuronal disorders is yet poorly understood and the potential ability of PACAP to modulate the related autophagic activation has never been significantly investigated. Hence, we explored the putative autophagy-modulating properties of PACAP in in vitro and in vivo models of PD, using the neurotoxic agents 1-methyl-4-phenylpyridinium (MPP(+)) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), respectively, to trigger alterations of DA neurons. In both models, following the toxin exposure, PACAP reduced the autophagic activity as evaluated by the production of LC3 II, the modulation of the p62 protein levels, and the formation of autophagic vacuoles. The ability of PACAP to inhibit autophagy was also observed in an in vitro cell assay by the blocking of the p62-sequestration activity produced with the autophagy inducer rapamycin. Thus, the results demonstrated that autophagy is induced in PD experimental models and that PACAP exhibits not only anti-apoptotic but also anti-autophagic properties.

  1. Cyclic adenosine 3',5'-monophosphate levels and activities of adenylate cyclase and cyclic adenosine 3',5'-monophosphate phosphodiesterase in Pseudomonas and Bacteroides.

    PubMed Central

    Siegel, L S; Hylemon, P B; Phibbs, P V

    1977-01-01

    A modified Gilman assay was used to determine the concentrations of cyclic adenosine 3',5'-monophosphate (cAMP) in rapidly filtered cells and in the culture filtrates of Pseudomonas aeruginosa, Escherichia coli K-12, and Bacteroides fragilis. In P. aeruginosa cultures, levels of cAMP in the filtrate increased with the culture absorbance (3.5 to 19.8 X 10(-9) M) but did not vary significantly with the carbon source used to support growth. Intracellular concentrations (0.8 to 3.2 X 10(-5) M) were substantially higher and did not vary appreciably during growth or with carbon source. Sodium cAMP (5 mM) failed to reverse the catabolite repression of inducible glucose-6-phosphate dehydrogenase (EC 1.1.1.49) synthesis caused by the addition of 10 mM succinate. Exogenous cAMP also had no discernible effect on the catabolite repression control of inducible mannitol dehydrogenase (EC 1.1.1.67). P. aeruginosa was found to contain both soluble cAMP phosphodiesterase (EC 3.1.4.17) and membrane-associated adenylate cyclase (EC 4.6.1.1) activity, and these were compared to the activities detected in crude extracts of E. coli. B. fragilis crude cell extracts contain neither of these enzyme activities, and little or no cAMP was detected in cells or culture filtrates of this anaerobic bacterium. PMID:187575

  2. Effect of structural analogs of butaclamol (a new antipsychotic drug) on striatal homovanillic acid and adenyl cyclase of olfactory tubercle in rats.

    PubMed

    Pugsley, T A; Merker, J; Lippman, W

    1976-08-01

    The 3-isopropyl (I), 3-cyclohexyl (II) and 3-phenyl (III) analogs of the new antipsychotic drug butaclamol, which contains a 3-tertiary butyl group, and their respective (+)-enantiomers, but not (-)-enantiomers, caused a dose related elevation of rat striatal homovanillic acid concentration, indicative of an increased dopamine (DA) turnover; droperidol also exhibited this activity. The order of activity of the (+)-enantiomers was (butaclamol) approximately II greater than I greater than III. A decrease in striatal DA was observed with (+)-I and (+)-III at the highest dose used, but not at one-half the dose. Each analog antagonized the DA-induced increase in adenyl cyclase (EC 4.6.1.1) activity of olfactory tubercle homogenates, the order of activity of the racemates (except for II) AND (+)-ENANTIOMERS BEING (BUTACLAMOL) APPROXIMATELY I greater than III greater than II. The (+)-enantiomers of butaclamol and analogs were two to four times more potent than their respective racemates, with (+)-butaclamol and (+)-I displaying activity generally equivalent to fluphenazine. The respective (-)-enantiomers were ineffective indicating a stereochemical specificity for DA-receptor blockade. Such analogs presented should be of value in elucidating dopaminergic mechansims.

  3. Changes in vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide and neuropeptide Y-ergic structures of the enteric nervous system in the carcinoma of the human large intestine.

    PubMed

    Godlewski, Janusz; Łakomy, Ireneusz Mirosław

    2010-01-01

    This investigation was aimed at immunohistochemical analysis of potential changes in the enteric nervous system caused by cancer of the large intestine. In this purpose, neurons and nerve fibers of intestinal plexuses containing neuropeptides: vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating polypeptide (PACAP) and neuropeptide Y (NPY), in pathologically changed part of the large intestine were microscpically observed and compared. Samples were taken from patients operated due to cancer of the sigmoid colon and rectum. The number of neurons and density of nerve fibres containing neuropeptides found in sections with cancer tissues were compared to those observed in sections from the uninvolved intestinal wall. Changes relating to reductions in the number of NPY-ergic neurons and density of nerve fibres in submucous and myenteric plexuses in the sections with cancer tissues (pathological sections) were statistically significant. A statistically similar presence of VIP-ergic and PACAP-ergic neurons in the submucosal and myenteric plexuses was observed in both the pathological and control sections. On the other hand, in the pathological sections, VIP-ergic nerve fibres in the myenteric plexuses and PACAP-ergic nerve fibres in the submucosal and myenteric plexuses were found to be less dense. Analysis revealed changes in pathologically affected part of the large intestine may caused disruption of proper intestinal function. Observed changes in the neural elements which are responsible for relaxation of the intestine may suggest dysfunction in the innervation of this part of the colon.

  4. Overexpression of adenylate cyclase-associated protein 1 may predict brain metastasis in non-small cell lung cancer.

    PubMed

    Xie, Shuan-Shuan; Tan, Min; Lin, Hai-Yan; Xu, Lei; Shen, Chang-Xing; Yuan, Qing; Song, Xiao-Lian; Wang, Chang-Hui

    2015-01-01

    This study was designed to establish a biomarker risk model for predicting brain metastasis (BM) in non-small cell lung cancer (NSCLC). The model comprises 120 cases of NSCLC that were treated and followed up for 4 years. The patients were divided into the BM (n=50) and non-BM (other visceral metastasis and those without recurrence) (n=70) groups. Immunohistochemical and western blot analyses were performed in metastatic tissues of NSCLC. Multivariate regression analysis was performed to correlate the immunoreactive cyclase-associated protein 1 (CAP1) signal with BM. Survival analyses were performed by using the Kaplan-Meier method. CAP1 protein content and immunoreactivity were significantly increased in BM specimens compared to other-metastatic specimens. The survival analysis revealed that CAP1 overexpression was significantly associated with survival (P<0.05). The ROC test suggested that the area under the curve was 73.33% (P<0.001; 95% CI, 63.5-83.2%). When P=0.466, the sensitivity and specificity reached 79.5 and 67.1%, respectively. These findings suggested that CAP1 is involved in the BM of NSCLC, and that elevated levels of CAP1 expression may indicate a poor prognosis for patients with BM. The CAP1 molecular model may be useful in the prediction of the risk of BM in NSCLC.

  5. Pituitary adenylate cyclase-activating polypeptide (PACAP) has a neuroprotective function in dopamine-based neurodegeneration in rat and snail parkinsonian models

    PubMed Central

    Kiss, Tibor; Jungling, Adel

    2017-01-01

    ABSTRACT Pituitary adenylate cyclase-activating polypeptide (PACAP) rescues dopaminergic neurons from neurodegeneration and improves motor changes induced by 6-hydroxy-dopamine (6-OHDA) in rat parkinsonian models. Recently, we investigated the molecular background of the neuroprotective effect of PACAP in dopamine (DA)-based neurodegeneration using rotenone-induced snail and 6-OHDA-induced rat models of Parkinson's disease. Behavioural activity, monoamine (DA and serotonin), metabolic enzyme (S-COMT, MB-COMT and MAO-B) and PARK7 protein concentrations were measured before and after PACAP treatment in both models. Locomotion and feeding activity were decreased in rotenone-treated snails, which corresponded well to findings obtained in 6-OHDA-induced rat experiments. PACAP was able to prevent the behavioural malfunctions caused by the toxins. Monoamine levels decreased in both models and the decreased DA level induced by toxins was attenuated by ∼50% in the PACAP-treated animals. In contrast, PACAP had no effect on the decreased serotonin (5HT) levels. S-COMT metabolic enzyme was also reduced but a protective effect of PACAP was not observed in either of the models. Following toxin treatment, a significant increase in MB-COMT was observed in both models and was restored to normal levels by PACAP. A decrease in PARK7 was also observed in both toxin-induced models; however, PACAP had a beneficial effect only on 6-OHDA-treated animals. The neuroprotective effect of PACAP in different animal models of Parkinson's disease is thus well correlated with neurotransmitter, enzyme and protein levels. The models successfully mimic several, but not all etiological properties of the disease, allowing us to study the mechanisms of neurodegeneration as well as testing new drugs. The rotenone and 6-OHDA rat and snail in vivo parkinsonian models offer an alternative method for investigation of the molecular mechanisms of neuroprotective agents, including PACAP. PMID:28067625

  6. Pituitary adenylate cyclase-activating polypeptide (PACAP) has a neuroprotective function in dopamine-based neurodegeneration in rat and snail parkinsonian models.

    PubMed

    Maasz, Gabor; Zrinyi, Zita; Reglodi, Dora; Petrovics, Dora; Rivnyak, Adam; Kiss, Tibor; Jungling, Adel; Tamas, Andrea; Pirger, Zsolt

    2017-02-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) rescues dopaminergic neurons from neurodegeneration and improves motor changes induced by 6-hydroxy-dopamine (6-OHDA) in rat parkinsonian models. Recently, we investigated the molecular background of the neuroprotective effect of PACAP in dopamine (DA)-based neurodegeneration using rotenone-induced snail and 6-OHDA-induced rat models of Parkinson's disease. Behavioural activity, monoamine (DA and serotonin), metabolic enzyme (S-COMT, MB-COMT and MAO-B) and PARK7 protein concentrations were measured before and after PACAP treatment in both models. Locomotion and feeding activity were decreased in rotenone-treated snails, which corresponded well to findings obtained in 6-OHDA-induced rat experiments. PACAP was able to prevent the behavioural malfunctions caused by the toxins. Monoamine levels decreased in both models and the decreased DA level induced by toxins was attenuated by ∼50% in the PACAP-treated animals. In contrast, PACAP had no effect on the decreased serotonin (5HT) levels. S-COMT metabolic enzyme was also reduced but a protective effect of PACAP was not observed in either of the models. Following toxin treatment, a significant increase in MB-COMT was observed in both models and was restored to normal levels by PACAP. A decrease in PARK7 was also observed in both toxin-induced models; however, PACAP had a beneficial effect only on 6-OHDA-treated animals. The neuroprotective effect of PACAP in different animal models of Parkinson's disease is thus well correlated with neurotransmitter, enzyme and protein levels. The models successfully mimic several, but not all etiological properties of the disease, allowing us to study the mechanisms of neurodegeneration as well as testing new drugs. The rotenone and 6-OHDA rat and snail in vivo parkinsonian models offer an alternative method for investigation of the molecular mechanisms of neuroprotective agents, including PACAP.

  7. Pituitary Adenylate Cyclase Activating Polypeptide, A Potential Therapeutic Agent for Diabetic Retinopathy in Rats: Focus on the Vertical Information Processing Pathway.

    PubMed

    Szabadfi, K; Reglodi, D; Szabo, A; Szalontai, B; Valasek, A; Setalo, Gy; Kiss, P; Tamas, A; Wilhelm, M; Gabriel, R

    2016-04-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurotrophic and neuroprotective peptide that has been shown to exert protective effects in different neuronal injuries, such as retinal degenerations. Diabetic retinopathy (DR), the most common complication of diabetes, affects the microvasculature and neuronal architecture of the retina. We have proven earlier that PACAP is also protective in a rat model of DR. In this study, streptozotocin-induced DR was treated with intravitreal PACAP administration in order to further analyze the synaptic structure and proteins of PACAP-treated diabetic retinas, primarily in the vertical information processing pathway. Streptozotocin-treated Wistar rats received intravitreal PACAP injection three times into the right eye 2 weeks after the induction of diabetes. Morphological and molecular biological (qRT-PCR; Western blot) methods were used to analyze retinal synapses (ribbons, conventional) and related structures. Electron microscopic analysis revealed that retinal pigment epithelium, the ribbon synapses and other synaptic profiles suffered alterations in diabetes. However, in PACAP-treated diabetic retinas more bipolar ribbon synapses were found intact in the inner plexiform layer than in DR animals. The ribbon synapse was marked with C-terminal binding protein 2/Bassoon and formed horseshoe-shape ribbons, which were more retained in PACAP-treated diabetic retinas than in DR rats. These results are supported by molecular biological data. The selective degeneration of related structures such as bipolar and ganglion cells could be ameliorated by PACAP treatment. In summary, intravitreal administration of PACAP may have therapeutic potential in streptozotocin-induced DR through maintaining synapse integrity in the vertical pathway.

  8. 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.

  9. Pharmacological, molecular and functional characterization of vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide receptors in the rat pineal gland.

    PubMed

    Simonneaux, V; Kienlen-Campard, P; Loeffler, J P; Basille, M; Gonzalez, B J; Vaudry, H; Robberecht, P; Pévet, P

    1998-08-01

    Melatonin secretion from the mammalian pineal gland is strongly stimulated by noradrenaline and also by vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP). Three types of receptors for VIP and PACAP have been characterized so far: VIP1/PACAP receptors and VIP2/PACAP receptors, which possess similar high affinities for VIP and PACAP, and PACAP1 receptors which exhibit a 100-1000-fold higher affinity for PACAP. The aim of the present study was to characterize the receptor subtype(s) mediating the stimulatory effects of VIP and PACAP on melatonin synthesis in the rat pineal gland. Autoradiographic studies showed that PACAP and VIP were equally potent in displacing binding of radioiodinated PACAP27 from pineal sections. Amplification of pineal complementary DNAs by polymerase chain reaction using specific primers for the different receptor subtypes revealed that all three receptor messenger RNAs are expressed and that VIP1/PACAP receptor messenger RNA was predominant over VIP2/PACAP receptor messenger RNA. In vitro, VIP and PACAP stimulated melatonin synthesis with similar high potency and the effect of the two peptides were not additive. The selective VIP1/PACAP receptor agonists [R16]chicken secretin (1-25) and [K15, R16, L27]VIP(1-7)/growth hormone releasing factor(8-27) were significantly more potent than the selective VIP2/PACAP receptor agonist RO 25-1553 in stimulating melatonin secretion. The stimulatory effects of VIP and PACAP were similarly inhibited by the VIP1/PACAP antagonist [acetyl-His1, D-Phe2, K15, R16, L27]VIP(3-7)/growth hormone releasing factor(8-27). These data strongly suggest that VIP and PACAP exert a stimulatory effect on melatonin synthesis mainly through activation of a pineal VIP1/PACAP receptor subtype.

  10. Spinal astrocytic activation contributes to both induction and maintenance of pituitary adenylate cyclase-activating polypeptide type 1 receptor-induced long-lasting mechanical allodynia in mice

    PubMed Central

    Yokai, Masafumi; Miyata, Atsuro

    2016-01-01

    Background Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are present in the spinal dorsal horn and dorsal root ganglia, suggesting an important role of PACAP–PACAP receptors signaling system in the modulation of spinal nociceptive transmission. We have previously reported that a single intrathecal injection of PACAP or a PACAP specific (PAC1) receptor selective agonist, maxadilan, in mice induced dose-dependent aversive behaviors, which lasted more than 30 min, and suggested that the maintenance of the nociceptive behaviors was associated with the spinal astrocytic activation. Results We found that a single intrathecal administration of PACAP or maxadilan also produced long-lasting hind paw mechanical allodynia, which persisted at least 84 days without affecting thermal nociceptive threshold. In contrast, intrathecal application of vasoactive intestinal polypeptide did not change mechanical threshold, and substance P, calcitonin gene-related peptide, or N-methyl-D-aspartate induced only transient mechanical allodynia, which disappeared within 21 days. Western blot and immunohistochemical analyses with an astrocytic marker, glial fibrillary acidic protein, revealed that the spinal PAC1 receptor stimulation caused sustained astrocytic activation, which also lasted more than 84 days. Intrathecal co-administration of L-α-aminoadipate, an astroglial toxin, with PACAP or maxadilan almost completely prevented the induction of the mechanical allodynia. Furthermore, intrathecal treatment of L-α-aminoadipate at 84 days after the PAC1 stimulation transiently reversed the mechanical allodynia accompanied by the reduction of glial fibrillary acidic protein expression level. Conclusion Our data suggest that spinal astrocytic activation triggered by the PAC1 receptor stimulation contributes to both induction and maintenance of the long-term mechanical allodynia. PMID:27175011

  11. Structural characterization by nuclear magnetic resonance spectroscopy of a genetically engineered high-affinity calmodulin-binding peptide derived from Bordetella pertussis adenylate cyclase.

    PubMed

    Munier, H; Bouhss, A; Gilles, A M; Palibroda, N; Bârzu, O; Mispelter, J; Craescu, C T

    1995-07-10

    This paper reports the solution conformation of a peptide (P196-267) derived from the calmodulin-binding domain of Bordetella pertussis adenylate cyclase. P196-267 corresponding to the protein fragment situated between amino acid residues 196-267 was overproduced by a recombinant Escherichia coli strain. Its affinity for calmodulin is only one order of magnitude lower (Kd = 2.4 nM) than that of the whole bacterial enzyme (Kd = 0.2 nM). The proton resonances of the NMR spectra of P196-267 were assigned using homonuclear two-dimensional techniques (double-quantum-filtered J-correlated spectroscopy, total correlation spectroscopy, and nuclear Overhauser enhancement spectroscopy) and a standard assignment procedure. Analysis of the nuclear Overhauser effect connectivities and the secondary shift distribution of C alpha protons along the sequence allowed us to identify the elements of regular secondary structure. The peptide is flexible in solution, being in equilibrium between random coil and helical structures. Two segments of 11 amino acids (situated between V215 and A225) and 15 amino acids (situated between L233 and A247) populate in a significant proportion the helix conformational state. The two helices can be considerably stabilized in a mixed solvent, trifluoroethanol/water (30/70), suggesting that the corresponding fragment in the intact protein assumes a similar secondary conformation. No elements of tertiary structure organization were detected by the present experiments. The conformational properties of the isolated calmodulin target fragment are discussed in relation with the available NMR and X-ray data on various peptides complexed to calmodulin.

  12. Ventilatory and cardiovascular actions of centrally and peripherally administered trout pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) in the unanaesthetized trout.

    PubMed

    Le Mével, J-C; Lancien, F; Mimassi, N; Conlon, J M

    2009-12-01

    In mammals, pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are involved in cardiovascular and respiratory regulation. Several studies have demonstrated the presence of PACAP, VIP and their receptors in various tissues of teleost fish, including the brain, but little is known about their respiratory and cardiovascular effects. The present study was undertaken to compare the central and peripheral actions of graded doses (25-100 pmol) of trout PACAP and trout VIP on ventilatory and cardiovascular variables in the unanaesthetized rainbow trout. Compared with vehicle, only intracerebroventricular injection of PACAP significantly (P<0.05) elevated the ventilation frequency and the ventilation amplitude, but both peptides significantly increased the total ventilation (total ventilation). However, the maximum hyperventilatory effect of PACAP was approximately 2.5-fold higher than the effect of VIP at the 100 pmol dose (PACAP, (total ventilation)=+5407+/-921 arbitrary units, a.u.; VIP, (total ventilation)=+2056+/-874 a.u.; means +/- s.e.m.). When injected centrally, only PACAP produced a significant increase in mean dorsal aortic blood pressure (P(DA)) (100 pmol: +21%) but neither peptide affected heart rate (f(H)). Intra-arterial injections of either PACAP or VIP were without effect on the ventilatory variables. PACAP was without significant action on P(DA) and f(H) while VIP significantly elevated P(DA) (100 pmol: +36%) without changing f(H). In conclusion, the selective central hyperventilatory actions of exogenously administered trout PACAP, and to a lesser extent VIP, suggest that the endogenous peptides may be implicated in important neuroregulatory functions related to the central control of ventilation in trout.

  13. [Differentially expressed genes identified in the main olfactory epithelium of mice with deficiency of adenylate cyclase 3 by using suppression subtractive hybridization approach].

    PubMed

    Zhenlong, Cao; Jiangye, Hao; Yanfen, Zhou; Zhe, Zhang; Zhihua, Ni; Yuanxiang, Hu; Weili, Liu; Yongchao, Li; Daniel, R Storm; Runlin, Z Ma; Zhenshan, Wang

    2014-06-01

    Adenylate cyclase 3 (AC3) is one of the major players in the olfactory signaling within the main olfactory epithelium (MOE) of mice. However, we are not ascertained whether deficiency of AC3 will lead to the differential expression of related genes in the MOE. Forward and reverse subtractive libraries were constructed by suppression subtractive hybridization (SSH) approach, with MOEs from AC3(-/-) and AC3(+/+) mice. These two libraries were primarily screened by Dot blot, differential expressed clones were sequenced and analyzed by bioinformatics, and differential expressed genes were verified by qRT-PCR. A total of 386 differentially expressed clones were picked out after Dot blot. The DNA sequences of 80 clones randomly selected were determined, and 62 clones were identified by blasting in GenBank. We found that 24 up-regulated clones were corresponded to genes of kcnk3, mapk7, megf11, and 38 down-regulated clones were corresponded to tmem88b, c-mip, skp1a, mlycd, etc. Their functions were annotated with Gene Ontology (GO) and found to be mainly focused on molecular binding, cell cycle, processes of biology and cells. Five genes (kcnk3, c-mip, mlycd, tmem88b and trappc5) were verified by qRT-PCR with individuals of AC3(+/+) and AC3(-/-) mice. The data indicate that kcnk3 gene is up-regulated significantly, increasing 1.27 folds compared to control mice, whereas c-mip, mlycd, tmem88b and trappc5 are down-regulated significantly, decreasing 20%, 7%, 32% and 29% compared to the AC3(+/+)mice. The functions of these genes are closely related with K(+) channels, cell differentiation, metabolism of fats, membrane transportation, and so on. It is tempting to speculate that these genes might work together with AC3 to orchestrate the olfactory transduction signaling in the MOE.

  14. Pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors are present and biochemically active in the central nervous system of the pond snail Lymnaea stagnalis.

    PubMed

    Pirger, Zsolt; Laszlo, Zita; Hiripi, Laszlo; Hernadi, Laszlo; Toth, Gabor; Lubics, Andrea; Reglodi, Dora; Kemenes, Gyorgy; Mark, Laszlo

    2010-11-01

    PACAP is a highly conserved adenylate cyclase (AC) activating polypeptide, which, along with its receptors (PAC1-R, VPAC1, and VPAC2), is expressed in both vertebrate and invertebrate nervous systems. In vertebrates, PACAP has been shown to be involved in associative learning, but it is not known if it plays a similar role in invertebrates. To prepare the way for a detailed investigation into the possible role of PACAP and its receptors in a suitable invertebrate model of learning and memory, here, we undertook a study of their expression and biochemical role in the central nervous system of the pond snail Lymnaea stagnalis. Lymnaea is one of the best established invertebrate model systems to study the molecular mechanisms of learning and memory, including the role of cyclic AMP-activated signaling mechanisms, which crucially depend on the learning-induced activation of AC. However, there was no information available on the expression of PACAP and its receptors in sensory structures and central ganglia of the Lymnaea nervous system known to be involved in associative learning or whether or not PACAP can actually activate AC in these ganglia. Here, using matrix-assisted laser desorption ionization time of flight (MALDI-TOF) and immunohistochemistry, we established the presence of PACAP-like peptides in the cerebral ganglia and the lip region of Lymnaea. The MALDI-TOF data indicated an identity with mammalian PACAP-27 and the presence of a squid-like PACAP-38 highly homologous to vertebrate PACAP-38. We also showed that PACAP, VIP, and maxadilan stimulated the synthesis of cAMP in Lymnaea cerebral ganglion homogenates and that this effect was blocked by the appropriate general and selective PACAP receptor antagonists.

  15. Testosterone regulates levels of cystic fibrosis transmembrane regulator, adenylate cyclase, and cAMP in the seminal vesicles of orchidectomized rats.

    PubMed

    Ramli, Nur Siti Khadijah; Giribabu, Nelli; Muniandy, Sekaran; Salleh, Naguib

    2016-01-15

    Secretions of chloride (Cl(-))- and bicarbonate (HCO3(-))-rich fluid by the seminal vesicles could involve cystic fibrosis transmembrane regulator (CFTR), which activity can be stimulated by cAMP generated from the reaction involving adenylate cyclase (AC). In this study, we investigated levels of CFTR, AC, and cAMP in the seminal vesicles under testosterone influence. Orchidectomized adult male rats received 7-day treatment with 125 or 250 μg/kg/day of testosterone with or without flutamide or finasteride. At the end of the treatment, animals were sacrificed and seminal vesicles were harvested for analyses of CFTR and AC protein expression level by Western blotting. Distribution of CFTR and AC in seminal vesicles was observed by immunohistochemistry. Levels of cAMP and dihydrotestosterone in seminal vesicle homogenates were measured by ELISA. Cystic fibrosis transmembrane regulator, AC, and cAMP levels increased with increasing doses of testosterone (P < 0.05 compared to nontreated orchidectomized rats). Cystic fibrosis transmembrane regulator and AC were expressed at the apical membrane of the epithelium lining the seminal vesicle lumen with higher expression levels observed in testosterone-treated rats than in non-treated orchidectomized rats (P < 0.05). The inhibitory effects of flutamide or finasteride on these parameters were greater in 250 μg/kg/day testosterone-treated rats than their effects in 125 μg/kg/day testosterone-treated rats. Higher dihydrotestosterone levels were observed in seminal vesicle homogenates after treatment with 250 μg/kg/day than with 125 μg/kg/day of testosterone (P < 0.05). Increased levels of CFTR, AC, and cAMP in seminal vesicles might contribute toward an increase in Cl(-) and HCO3(-) concentrations in the seminal fluid as reported under testosterone influence.

  16. Chronic ghrelin treatment reduced photophobia and anxiety-like behaviors in nitroglycerin- induced migraine: role of pituitary adenylate cyclase-activating polypeptide.

    PubMed

    Farajdokht, Fereshteh; Babri, Shirin; Karimi, Pouran; Alipour, Mohammad Reza; Bughchechi, Ramin; Mohaddes, Gisou

    2017-03-01

    Chronic migraine is a debilitating disorder that has a significant impact on patients and society. Nearly all migraineurs frequently reported light sensitivity during a headache attack. Pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in the activation of trigeminal system and migraine pain. To identify the effect of chronic ghrelin treatment on endogenous PACAP and associated symptoms of migraine, an experimental chronic migraine model was induced by intermittent intraperitoneal (i.p) injection of nitroglycerin (NTG). Photophobia and anxiety-like behaviors were determined in the modified elevated plus maze on days 2, 4, 6, 8, and 10 and in the light/dark box on days 3, 5, 7, 9, and 11. Blood levels of PACAP and cortisol were assessed by enzyme-linked immunosorbent (ELISA) kits. Chronic injection of NTG evoked photophobia and anxiety-like behaviors and treatment with ghrelin (150 μg/kg) for 11 days effectively attenuated photophobia and anxiety-like behaviors in the both paradigms. We further found that NTG increased the blood levels of PACAP and cortisol, which was significantly reduced by ghrelin treatment. Additionally, staining with Hematoxylin and Eosin (H&E) revealed that ghrelin reduced NTG-induced increase in the number of satellite glial cells in the trigeminal ganglion. Furthermore, for the first time we showed that repeated administrations of NTG increased white blood cell (WBC) counts and mean platelet volume (MPV), and decreased platelet counts. These results indicated that ghrelin decreased migraine associated symptoms possibly through attenuating endogenous PACAP and cortisol levels. Therefore, ghrelin may hold therapeutic potentialities in managing the chronic migraine.

  17. Pituitary adenylate cyclase-activating polypeptide (PACAP) inhibits the slow afterhyperpolarizing current sIAHP in CA1 pyramidal neurons by activating multiple signaling pathways.

    PubMed

    Taylor, Ruth D T; Madsen, Marita Grønning; Krause, Michael; Sampedro-Castañeda, Marisol; Stocker, Martin; Pedarzani, Paola

    2014-01-01

    The slow afterhyperpolarizing current (sIAHP ) is a calcium-dependent potassium current that underlies the late phase of spike frequency adaptation in hippocampal and neocortical neurons. sIAHP is a well-known target of modulation by several neurotransmitters acting via the cyclic AMP (cAMP) and protein kinase A (PKA)-dependent pathway. The neuropeptide pituitary adenylate cyclase activating peptide (PACAP) and its receptors are present in the hippocampal formation. In this study we have investigated the effect of PACAP on the sIAHP and the signal transduction pathway used to modulate intrinsic excitability of hippocampal pyramidal neurons. We show that PACAP inhibits the sIAHP , resulting in a decrease of spike frequency adaptation, in rat CA1 pyramidal cells. The suppression of sIAHP by PACAP is mediated by PAC1 and VPAC1 receptors. Inhibition of PKA reduced the effect of PACAP on sIAHP, suggesting that PACAP exerts part of its inhibitory effect on sIAHP by increasing cAMP and activating PKA. The suppression of sIAHP by PACAP was also strongly hindered by the inhibition of p38 MAP kinase (p38 MAPK). Concomitant inhibition of PKA and p38 MAPK indicates that these two kinases act in a sequential manner in the same pathway leading to the suppression of sIAHP. Conversely, protein kinase C is not part of the signal transduction pathway used by PACAP to inhibit sIAHP in CA1 neurons. Our results show that PACAP enhances the excitability of CA1 pyramidal neurons by inhibiting the sIAHP through the activation of multiple signaling pathways, most prominently cAMP/PKA and p38 MAPK. Our findings disclose a novel modulatory action of p38 MAPK on intrinsic excitability and the sIAHP, underscoring the role of this current as a neuromodulatory hub regulated by multiple protein kinases in cortical neurons.

  18. Role for the beta-adrenoceptor-coupled adenylate cyclase in the ontogenetic subsensitivity to isoproterenol in the embryonic chick ventricle

    SciTech Connect

    Smith, C.J.

    1985-01-01

    Isoproterenol (ISO) increases contractility and cyclic AMP content in ventricles of embryonic and hatched chicks. A transient decrease in beta-agonist sensitivity for both effects is seen in 18 day embryos (10E). Beta-adrenoceptor-coupled adenylate cylase (AC) and receptor binding were characterized in 14,000xg particulates and purified membranes from the ventricles of 10-11E, 17-19E and week-old chicks (5-6H). In crude particulates, the K/sub act/ for ISO (+100 ..mu..M Gpp(NH)p)-stimulated AC is greatest in the 17-19E. Maximal (ISO + Gpp(NH)p)-AC of the 11E is two-fold greater and NaF-AC is 30% greater than those of the 17-19E and 5-6 H. All age groups have comparable catalytic AC. All age groups have comparable K/sub d/'s for /sup 3/H-dihydroalprenolol (5-11 nM), while the 18E has 40% fewer receptors than the 11E and 5-6H. In particulates or membranes, K/sub act/ values for Gpp(NH)p, NaF, MnCl> and forskolin are unchanged with age. In membranes, K/sub act/ values for ISO plus guanine nucleotide (G) and maximal (ISO + G)-AC are similar in all ages. The net effect of ISO ((ISO + G) minus G) is least while that of G (G minus basal) is greatest in the 18E. Whereas /sup 32/P-labeling of a 42 kd protein by cholera toxin is lowest (25% decrease) in particulates of the 18E, labeling of a 39-41 kd doublet by pertussis toxin decreases continuously (by 50%) with age. All age groups have comparable K/sub d/'s (10-13 pM) for (/sup 125/I)-cyanopindolol (CYP). These data indicate that a transient decrease in receptor number and receptor-N/sub s/ (guanine nucleotide-sensitive) coupling in the 18E contribute to the subsensitivity to beta-agonist.

  19. Direct interaction between the catalytic subunit of the calmodulin-sensitive adenylate cyclase from bovine brain with /sup 125/I-labeled wheat germ agglutinin and /sup 125/I-labeled calmodulin

    SciTech Connect

    Minocherhomjee, A.M.; Selfe, S.; Flowers, N.J.; Storm, D.R.

    1987-07-14

    A calmodulin-sensitive adenylate cyclase has been purified to apparent homogeneity from bovine cerebral cortex using calmodulin-Sepharose followed by forskolin-Sepharose and wheat germ agglutinin-Sepharose. The final product appeared as one major polypeptide of approximately 135,000 daltons on sodium dodecyl sulfate-polyacrylamide gels. This polypeptide was a major component of the protein purified through calmodulin-Sepharose. The catalytic subunit was stimulated 3-4-fold by calmodulin (CaM) with a turnover number greater than 1000 min/sup -1/ and was directly inhibited by adenosine. The catalytic subunit of the enzyme interacted directly with /sup 125/I-CaM on a sodium dodecyl sulfate-polyacrylamide gel overlay system, and this interaction was Ca/sup 2 +/ concentration dependent. In addition, the catalytic subunit was shown to directly bind /sup 125/I-labeled wheat germ agglutinin using a sodium dodecyl sulfate-polyacrylamide gel overlay technique, and N-acetylglucosamine inhibited binding of the lectin to the catalytic subunit. Calmodulin did not inhibit binding of wheat germ agglutinin to the catalytic subunit, and the binding of calmodulin was unaffected by wheat germ agglutinin. These data illustrate that the catalytic subunit of the calmodulin-sensitive adenylate cyclase is a glycoprotein which interacts directly with calmodulin and that adenosine can inhibit the enzyme without intervening receptors or G coupling proteins. It is concluded that the catalytic subunit of adenylate cyclase is a transmembrane protein with a domain accessible from the outer surface of the cell.

  20. Increased sensitivity in the interaction of the dopaminergic/adenosinergic system at the level of the adenylate cyclase activity in the striatum of the "weaver" mouse.

    PubMed

    K, Botsakis; V, Tondikidou; N, Panagopoulos; M, Margariti; N, Matsokis; F, Angelatou

    2016-10-01

    The specific antagonistic interaction between dopamine D1 and adenosine A1 receptors (D1/A1), as well as between dopamine D2 and adenosine A2a receptors (D2/A2a) exist not only at the receptor/receptor level, but also at the level of the secondary messengers. In this study, we examined the possible changes in these interactions at the level of cAMP formation in membrane preparation from "weaver" mouse striatum (a genetic model of Parkinson disease), by using specific agonists of these receptors. We also examined in the striatum of the "weaver" mouse the interaction between D1 and D2 dopamine receptors. Our results showed that in the striatum of "weaver" mice: a) the cAMP synthesis induced by D1 receptor activation (SKF 38393), was significantly reduced compared to control mice, while A1 receptor activation (L-PIA) leaded to a more intense inhibition of the D1-induced cAMP-formation compared to the controls, b) the cAMP synthesis which was induced by A2a receptor activation (CGS 21680), was significantly increased compared to the control mice. The specific D2 receptor agonist Quinpirole, added in low concentrations, caused a significant reduction of the A2a-induced cAMP formation, which was not observed in the control mouse. Furthermore, the D1 receptor induced cAMP synthesis was significantly higher in control compared to "weaver" striatum, which was more efficiently downregulated by D2 receptor agonist Quinpirole. These results suggest that the sensitivity to D1 and A2a receptor agonists is altered and that the interaction between D1/A1 and D2/A2a receptors is enhanced in the striatum of the "weaver" mutation, while an uncoupling between D1 and D2 receptors was observed. Since the adenylate cyclase basal activity did not differ between "weaver" and control striatum, the above-mentioned changes seem to be due to alterations in the function of the adenosine/dopamine receptors and their coupling to the G-proteins.

  1. Racemic Salsolinol and its Enantiomers Act as Agonists of the μ-Opioid Receptor by Activating the Gi Protein-Adenylate Cyclase Pathway.

    PubMed

    Berríos-Cárcamo, Pablo; Quintanilla, María E; Herrera-Marschitz, Mario; Vasiliou, Vasilis; Zapata-Torres, Gerald; Rivera-Meza, Mario

    2016-01-01

    Background: Several studies have shown that the ethanol-derived metabolite salsolinol (SAL) can activate the mesolimbic system, suggesting that SAL is the active molecule mediating the rewarding effects of ethanol. In vitro and in vivo studies suggest that SAL exerts its action on neuron excitability through a mechanism involving opioid neurotransmission. However, there is no direct pharmacologic evidence showing that SAL activates opioid receptors. Methods: The ability of racemic (R/S)-SAL, and its stereoisomers (R)-SAL and (S)-SAL, to activate the μ-opioid receptor was tested in cell-based (light-emitting) receptor assays. To further characterizing the interaction of SAL stereoisomers with the μ-opioid receptor, a molecular docking study was performed using the crystal structure of the μ-opioid receptor. Results: This study shows that SAL activates the μ-opioid receptor by the classical G protein-adenylate cyclase pathway with an half-maximal effective concentration (EC50) of 2 × 10(-5) M. The agonist action of SAL was fully blocked by the μ-opioid antagonist naltrexone. The EC50 for the purified stereoisomers (R)-SAL and (S)-SAL were 6 × 10(-4) M and 9 × 10(-6) M respectively. It was found that the action of racemic SAL on the μ-opioid receptor did not promote the recruitment of β-arrestin. Molecular docking studies showed that the interaction of (R)- and (S)-SAL with the μ-opioid receptor is similar to that predicted for the agonist morphine. Conclusions: It is shown that (R)-SAL and (S)-SAL are agonists of the μ-opioid receptor. (S)-SAL is a more potent agonist than the (R)-SAL stereoisomer. In silico analysis predicts a morphine-like interaction between (R)- and (S)-SAL with the μ-opioid receptor. These results suggest that an opioid action of SAL or its enantiomers is involved in the rewarding effects of ethanol.

  2. Racemic Salsolinol and its Enantiomers Act as Agonists of the μ-Opioid Receptor by Activating the Gi Protein-Adenylate Cyclase Pathway

    PubMed Central

    Berríos-Cárcamo, Pablo; Quintanilla, María E.; Herrera-Marschitz, Mario; Vasiliou, Vasilis; Zapata-Torres, Gerald; Rivera-Meza, Mario

    2017-01-01

    Background: Several studies have shown that the ethanol-derived metabolite salsolinol (SAL) can activate the mesolimbic system, suggesting that SAL is the active molecule mediating the rewarding effects of ethanol. In vitro and in vivo studies suggest that SAL exerts its action on neuron excitability through a mechanism involving opioid neurotransmission. However, there is no direct pharmacologic evidence showing that SAL activates opioid receptors. Methods: The ability of racemic (R/S)-SAL, and its stereoisomers (R)-SAL and (S)-SAL, to activate the μ-opioid receptor was tested in cell-based (light-emitting) receptor assays. To further characterizing the interaction of SAL stereoisomers with the μ-opioid receptor, a molecular docking study was performed using the crystal structure of the μ-opioid receptor. Results: This study shows that SAL activates the μ-opioid receptor by the classical G protein-adenylate cyclase pathway with an half-maximal effective concentration (EC50) of 2 × 10−5 M. The agonist action of SAL was fully blocked by the μ-opioid antagonist naltrexone. The EC50 for the purified stereoisomers (R)-SAL and (S)-SAL were 6 × 10−4 M and 9 × 10−6 M respectively. It was found that the action of racemic SAL on the μ-opioid receptor did not promote the recruitment of β-arrestin. Molecular docking studies showed that the interaction of (R)- and (S)-SAL with the μ-opioid receptor is similar to that predicted for the agonist morphine. Conclusions: It is shown that (R)-SAL and (S)-SAL are agonists of the μ-opioid receptor. (S)-SAL is a more potent agonist than the (R)-SAL stereoisomer. In silico analysis predicts a morphine-like interaction between (R)- and (S)-SAL with the μ-opioid receptor. These results suggest that an opioid action of SAL or its enantiomers is involved in the rewarding effects of ethanol. PMID:28167903

  3. Pituitary adenylate cyclase-activating polypeptide (PACAP) inhibits the slow afterhyperpolarizing current sIAHP in CA1 pyramidal neurons by activating multiple signaling pathways

    PubMed Central

    Taylor, Ruth DT; Madsen, Marita Grønning; Krause, Michael; Sampedro-Castañeda, Marisol; Stocker, Martin; Pedarzani, Paola

    2014-01-01

    The slow afterhyperpolarizing current (sIAHP) is a calcium-dependent potassium current that underlies the late phase of spike frequency adaptation in hippocampal and neocortical neurons. sIAHP is a well-known target of modulation by several neurotransmitters acting via the cyclic AMP (cAMP) and protein kinase A (PKA)-dependent pathway. The neuropeptide pituitary adenylate cyclase activating peptide (PACAP) and its receptors are present in the hippocampal formation. In this study we have investigated the effect of PACAP on the sIAHP and the signal transduction pathway used to modulate intrinsic excitability of hippocampal pyramidal neurons. We show that PACAP inhibits the sIAHP, resulting in a decrease of spike frequency adaptation, in rat CA1 pyramidal cells. The suppression of sIAHP by PACAP is mediated by PAC1 and VPAC1 receptors. Inhibition of PKA reduced the effect of PACAP on sIAHP, suggesting that PACAP exerts part of its inhibitory effect on sIAHP by increasing cAMP and activating PKA. The suppression of sIAHP by PACAP was also strongly hindered by the inhibition of p38 MAP kinase (p38 MAPK). Concomitant inhibition of PKA and p38 MAPK indicates that these two kinases act in a sequential manner in the same pathway leading to the suppression of sIAHP. Conversely, protein kinase C is not part of the signal transduction pathway used by PACAP to inhibit sIAHP in CA1 neurons. Our results show that PACAP enhances the excitability of CA1 pyramidal neurons by inhibiting the sIAHP through the activation of multiple signaling pathways, most prominently cAMP/PKA and p38 MAPK. Our findings disclose a novel modulatory action of p38 MAPK on intrinsic excitability and the sIAHP, underscoring the role of this current as a neuromodulatory hub regulated by multiple protein kinases in cortical neurons. © 2013 The Authors. Hippocampus Published by Wiley Periodicals, Inc. PMID:23996525

  4. Characterizations of a synthetic pituitary adenylate cyclase-activating polypeptide analog displaying potent neuroprotective activity and reduced in vivo cardiovascular side effects in a Parkinson's disease model.

    PubMed

    Lamine, Asma; Létourneau, Myriam; Doan, Ngoc Duc; Maucotel, Julie; Couvineau, Alain; Vaudry, Hubert; Chatenet, David; Vaudry, David; Fournier, Alain

    2016-09-01

    Parkinson's disease (PD) is characterized by a steady loss of dopamine neurons through apoptotic, inflammatory and oxidative stress processes. In that line of view, the pituitary adenylate cyclase-activating polypeptide (PACAP), with its ability to cross the blood-brain barrier and its anti-apoptotic, anti-inflammatory and anti-oxidative properties, has proven to offer potent neuroprotection in various PD models. Nonetheless, its peripheral actions, paired with low metabolic stability, hampered its clinical use. We have developed Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27) as an improved PACAP-derived neuroprotective compound. In vitro, this analog stimulated cAMP production, maintained mitochondrial potential and protected SH-SY5Y neuroblastoma cells from 1-methyl-4-phenylpyridinium (MPP(+)) toxicity, as potently as PACAP. Furthermore, contrasting with PACAP, it is stable in human plasma and against dipeptidyl peptidase IV activity. When injected intravenously to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, PACAP and Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27) restored tyrosine hydoxylase expression into the substantia nigra and modulated the inflammatory response. Albeit falls of mean arterial pressure (MAP) were observed with both PACAP- and Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27)-treated mice, the intensity of the decrease as well as its duration were significantly less marked after iv injections of the analog than after those of the native polypeptide. Moreover, no significant changes in heart rate were measured with the animals for both compounds. Thus, Ac-[Phe(pI)(6), Nle(17)]PACAP(1-27) appears as a promising lead molecule for the development of PACAP-derived drugs potentially useful for the treatment of PD or other neurodegenerative diseases.

  5. Adenylate cyclase and the cyclic AMP receptor protein modulate stress resistance and virulence capacity of uropathogenic Escherichia coli.

    PubMed

    Donovan, Grant T; Norton, J Paul; Bower, Jean M; Mulvey, Matthew A

    2013-01-01

    In many bacteria, the second messenger cyclic AMP (cAMP) interacts with the transcription factor cAMP receptor protein (CRP), forming active cAMP-CRP complexes that can control a multitude of cellular activities, including expanded carbon source utilization, stress response pathways, and virulence. Here, we assessed the role of cAMP-CRP as a regulator of stress resistance and virulence in uropathogenic Escherichia coli (UPEC), the principal cause of urinary tract infections worldwide. Deletion of genes encoding either CRP or CyaA, the enzyme responsible for cAMP synthesis, attenuates the ability of UPEC to colonize the bladder in a mouse infection model, dependent on intact innate host defenses. UPEC mutants lacking cAMP-CRP grow normally in the presence of glucose but are unable to utilize alternate carbon sources like amino acids, the primary nutrients available to UPEC within the urinary tract. Relative to the wild-type UPEC isolate, the cyaA and crp deletion mutants are sensitive to nitrosative stress and the superoxide generator methyl viologen but remarkably resistant to hydrogen peroxide (H(2)O(2)) and acid stress. In the mutant strains, H(2)O(2) resistance correlates with elevated catalase activity attributable in part to enhanced translation of the alternate sigma factor RpoS. Acid resistance was promoted by both RpoS-independent and RpoS-dependent mechanisms, including expression of the RpoS-regulated DNA-binding ferritin-like protein Dps. We conclude that balanced input from many cAMP-CRP-responsive elements, including RpoS, is critical to the ability of UPEC to handle the nutrient limitations and severe environmental stresses present within the mammalian urinary tract.

  6. Central pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) decrease the baroreflex sensitivity in trout.

    PubMed

    Lancien, Frédéric; Mimassi, Nagi; Conlon, J Michael; Le Mével, Jean-Claude

    2011-04-01

    Although PACAP and VIP exert diverse actions on heart and blood vessels along the vertebrate phylum, no information is currently available concerning the potential role of these peptides on the regulation of the baroreflex response, a major mechanism for blood pressure homeostasis. Consequently, the goal of this study was to examine in our experimental model, the unanesthetized rainbow trout Oncorhynchus mykiss, whether PACAP and VIP are involved in the regulation of the cardiac baroreflex sensitivity (BRS). Cross-spectral analysis techniques using a fast Fourier transform algorithm were employed to calculate the coherence, phase and gain of the transfer function between spontaneous fluctuations of systolic arterial blood pressure and R-R intervals of the electrocardiogram. The BRS was estimated as the mean of the gain of the transfer function when the coherence between the two signals was high and the phase negative. Compared with vehicle, intracerebroventricular (i.c.v.) injections of trout PACAP-27 and trout VIP (25-100 pmol) dose-dependently reduced the cardiac BRS to the same extent with a threshold dose of 50 pmol for a significant effect. When injected intra-arterially at the same doses as for i.c.v. injections, only the highest dose of VIP (100 pmol) significantly attenuated the BRS. These results suggest that the endogenous peptides PACAP and VIP might be implicated in the central control of cardiac baroreflex functions in trout.

  7. Roles for pituitary adenylate cyclase-activating peptide (PACAP) expression and signaling in the bed nucleus of the stria terminalis (BNST) in mediating the behavioral consequences of chronic stress.

    PubMed

    Hammack, Sayamwong E; Roman, Carolyn W; Lezak, Kimberly R; Kocho-Shellenberg, Margaret; Grimmig, Bethany; Falls, William A; Braas, Karen; May, Victor

    2010-11-01

    Anxiety disorders are frequently long-lasting and debilitating for more than 40 million American adults. Although stressor exposure plays an important role in the etiology of some anxiety disorders, the mechanisms by which exposure to stressful stimuli alters central circuits that mediate anxiety-like emotional behavior are still unknown. Substantial evidence has implicated regions of the central extended amygdala, including the bed nucleus of the stria terminalis (BNST) and the central nucleus of the amygdala as critical structures mediating fear- and anxiety-like behavior in both humans and animals. These areas organize coordinated fear- and anxiety-like behavioral responses as well as peripheral stress responding to threats via direct and indirect projections to the paraventricular nucleus of the hypothalamus and brainstem regions (Walker et al. Eur J Pharmacol 463:199-216, 2003, Prog Neuropsychopharmacol Biol Psychiatry 33(8):1291-1308, 2009; Ulrich-Lai and Herman Nat Rev Neurosci 10:397-409, 2009). In particular, the BNST has been argued to mediate these central and peripheral responses when the perceived threat is of long duration (Waddell et al. Behav Neurosci 120:324-336, 2006) and/or when the anxiety-like response is sustained (Walker and Davis Brain Struct Funct 213:29-42, 2008); hence, the BNST may mediate pathological anxiety-like states that result from exposure to chronic stress. Indeed, chronic stress paradigms result in enhanced BNST neuroplasticity that has been associated with pathological anxiety-like states (Vyas et al. Brain Res 965:290-294, 2003; Pego et al. Eur J Neurosci 27:1503-1516, 2008). Here we review evidence that suggests that pituitary adenylate cyclase-activating polypeptide (PACAP) and corticotropin-releasing hormone (CRH) work together to modulate BNST function and increase anxiety-like behavior. Moreover, we have shown that BNST PACAP as well as its cognate PAC1 receptor is substantially upregulated following chronic stress

  8. Follicle-stimulating hormone receptor-mediated uptake of sup 45 Ca sup 2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase

    SciTech Connect

    Grasso, P.; Reichert, L.E. Jr. )

    1990-08-01

    We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake. Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis. PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx. The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged. Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC. Our data further suggest that the FSH receptor itself may function as a calcium channel.

  9. Site-directed mutagenesis of human beta-adrenergic receptors: substitution of aspartic acid-130 by asparagine produces a receptor with high-affinity agonist binding that is uncoupled from adenylate cyclase.

    PubMed Central

    Fraser, C M; Chung, F Z; Wang, C D; Venter, J C

    1988-01-01

    By using oligonucleotide-directed mutagenesis, we have produced a point mutation (guanine to adenine) at nucleotide 388 of the gene for human beta-adrenergic receptor (beta AR) that results in a substitution of asparagine for the highly conserved aspartic acid at position 130 in the putative third transmembrane domain of the human beta AR ([Asn130]beta AR). We have examined the functional significance of this mutation in B-82 cells continuously expressing the mutant [Asn130]beta AR. The mutant [Asn130]beta AR displayed normal antagonist binding but unusually high-affinity agonist binding (5- to 10-fold higher than wild-type beta AR), consistent with a single class of high-affinity binding sites. The mutant beta AR displayed guanine nucleotide-sensitive changes in agonist affinity (3- to 5-fold shift) implying an interaction between the beta AR and the stimulatory guanine nucleotide-binding regulatory protein; however, the ability of guanine nucleotides to alter agonist affinity was attenuated. Addition of saturating concentrations of isoproterenol to cell cultures expressing mutant [Asn130]-beta ARs had no effect on intracellular levels of cAMP, indicating that the mutant beta AR is unable to affect stimulation of adenylate cyclase. These results indicate that substitution of the aspartic acid with asparagine at residue 130 of the human beta AR dissociates the well-characterized guanine nucleotide effects on agonist affinity from those on activation of the stimulatory guanine nucleotide-binding regulatory protein and adenylate cyclase and suggests the existence of two distinct counterions for the amine portion of catecholamines that are associated with high- and low-affinity agonist binding states of beta AR. Images PMID:2840663

  10. Identification of bacterial guanylate cyclases.

    PubMed

    Ryu, Min-Hyung; Youn, Hwan; Kang, In-Hye; Gomelsky, Mark

    2015-05-01

    The ability of bacteria to use cGMP as a second messenger has been controversial for decades. Recently, nucleotide cyclases from Rhodospirillum centenum, GcyA, and Xanthomonas campestris, GuaX, have been shown to possess guanylate cyclase activities. Enzymatic activities of these guanylate cyclases measured in vitro were low, which makes interpretation of the assays ambiguous. Protein sequence analysis at present is insufficient to distinguish between bacterial adenylate and guanylate cyclases, both of which belong to nucleotide cyclases of type III. We developed a simple method for discriminating between guanylate and adenylate cyclase activities in a physiologically relevant bacterial system. The method relies on the use of a mutant cAMP receptor protein, CRPG , constructed here. While wild-type CRP is activated exclusively by cAMP, CRPG can be activated by either cAMP or cGMP. Using CRP- and CRPG -dependent lacZ expression in two E. coli strains, we verified that R. centenum GcyA and X. campestris GuaX have primarily guanylate cyclase activities. Among two other bacterial nucleotide cyclases tested, one, GuaA from Azospillrillum sp. B510, proved to have guanylate cyclase activity, while the other one, Bradyrhizobium japonicum CyaA, turned out to function as an adenylate cyclase. The results obtained with this reporter system were in excellent agreement with direct measurements of cyclic nucleotides secreted by E. coli expressing nucleotide cyclase genes. The simple genetic screen developed here is expected to facilitate identification of bacterial guanylate cyclases and engineering of guanylate cyclases with desired properties.

  11. Examination of calcium-binding protein expression in the inner ear of wild-type, heterozygous and homozygous pituitary adenylate cyclase-activating polypeptide (PACAP)-knockout mice in kanamycin-induced ototoxicity.

    PubMed

    Nemeth, A; Szabadfi, K; Fulop, B; Reglodi, D; Kiss, P; Farkas, J; Szalontai, B; Gabriel, R; Hashimoto, H; Tamas, A

    2014-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with diverse biological effects. It also occurs and exerts protective effects in sensory organs; however, little is known about its effects in the auditory system. Recently, we have shown that PACAP protects cochlear cells against oxidative-stress-induced apoptosis and homozygous PACAP-deficient animals show stronger expression of Ca(2+)-binding proteins in the hair cells of the inner ear, but there are no data about the consequences of the lack of endogenous PACAP in different ototoxic insults such as aminoglycoside-induced toxicity. In this study, we examined the effect of kanamycin treatment on Ca(2+)-binding protein expression in hair cells of wild-type, heterozygous and homozygous PACAP-deficient mice. We treated 5-day-old mice with kanamycin, and 2 days later, we examined the Ca(2+)-binding protein expression of the hair cells with immunohistochemistry. We found stronger expression of Ca(2+)-binding proteins in the hair cells of control heterozygous and homozygous PACAP-deficient mice compared with wild-type animals. Kanamycin induced a significant increase in Ca(2+)-binding protein expression in wild-type and heterozygous PACAP-deficient mice, but the baseline higher expression in homozygous PACAP-deficient mice did not show further changes after the treatment. Elevated endolymphatic Ca(2+) is deleterious for the cochlear function, against which the high concentration of Ca(2+)-buffers in hair cells may protect. Meanwhile, the increased immunoreactivity of Ca(2+)-binding proteins in the absence of PACAP provide further evidence for the important protective role of PACAP in ototoxicity, but further investigations are necessary to examine the exact role of endogenous PACAP in ototoxic insults.

  12. The role of activation of the 5-HT1A receptor and adenylate cyclase in the antidepressant-like effect of YL-0919, a dual 5-HT1A agonist and selective serotonin reuptake inhibitor.

    PubMed

    Qin, Juan-Juan; Chen, Hong-Xia; Zhao, Nan; Yuan, Li; Zhang, You-Zhi; Yang, Ri-Fang; Zhang, Li-Ming; Li, Yun-Feng

    2014-10-17

    This study aimed to explore the possible mechanisms underlying the antidepressant-like effect of YL-0919, a novel antidepressant candidate with dual activity as a 5-HT1A receptor agonist and a selective serotonin reuptake inhibitor. The animal models commonly used to evaluate potential antidepressants, i.e., tail suspension (TST) in mice and forced swimming test (FST) in mice were used to evaluate the antidepressant effect of YL-0919. The activity of adenylate cyclase (AC) on the synaptic membrane was determined by the homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. The results indicated that YL-0919 (1.25-2.5mg/kg, i.g.) significantly decreased the immobility time in both the tail suspension test and the forced swim test in a dose-dependent manner, demonstrating the antidepressant-like effect of YL-0919. Furthermore, this effect was completely antagonized by the co-administration of WAY-100635 (0.3mg/kg, s.c.), a 5-HT1A selective antagonist. YL-0919 (10(-9)-10(-5)mol/L) was also shown to activate AC in vitro in a dose-dependent manner in synaptic membranes extracted from the rat prefrontal cortex, and this effect (10(-7)-10(-5)mol/L) was antagonized by WAY-100635 (10(-7)mol/L). Finally, the antidepressant-like effect of YL-0919 (2.5mg/kg, i.g.) was also blocked by the co-administration of H-89 (3 μg/site, i.c.v.), a protein kinase A (PKA) selective inhibitor. These results indicate that the activation of 5-HT1A receptors and the subsequent activation of the AC-cAMP-PKA signaling pathway in the frontal cortex play a critical role in the antidepressant-like effect of YL-0919.

  13. The human vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor 1 (VPAC1) promoter: characterization and role in receptor expression during enterocytic differentiation of the colon cancer cell line Caco-2Cl.20.

    PubMed Central

    Couvineau, A; Maoret, J J; Rouyer-Fessard, C; Carrero, I; Laburthe, M

    2000-01-01

    The basic organization of the human vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor (VPAC) 1 promoter was investigated after cloning the 5'-flanking region (1.4 kb) of the VPAC1 gene from a human genomic library. Subsequent functional analysis of various deletions of the 5'-flanking sequence, subcloned upstream of a luciferase reporter gene, was carried out in HT-29 cells. The minimal promoter region identified encompasses the -205/+76 sequence and contains a crucial CCAAT box (-182/-178) and a GC-rich sequence. Moreover a region (-1348/-933) containing a silencer element was identified. We previously showed that the expression of the VPAC1 receptor binding site is strictly dependent upon the enterocytic differentiation of human colon cancer Caco-2 cells [Laburthe, Rousset, Rouyer-Fessard, Couvineau, Chantret, Chevalier and Zweibaum (1987) J. Biol. Chem. 262, 10180-10184]. In the present study we show that VPAC1 mRNA increases dramatically when Caco-2Cl.20 cells differentiate, as measured by RNase protection assays and reverse transcriptase-PCR. A single transcript species of 3 kb is detected in differentiated cells by Northern-blot analysis. Accumulation of VPAC1 receptor mRNA is due to a 5-fold increase of transcription rate (run-on assay) without a change in mRNA half-life (9 h). Stable transfections of various constructs in Caco-2Cl.20 cells and subsequent analysis of reporter gene expression, during the enterocytic differentiation process over 25 days of culture, further indicated that the -254/+76 5'-flanking sequence is endowed with the regulatory element(s) necessary for transcriptional regulation of VPAC1 during differentiation. Altogether, these observations provide the first characterization of the basic organization of the human VPAC1 gene promoter and unravel the crucial role of a short promoter sequence in the strict transcriptional control of VPAC1 expression during differentiation of human colon cancer Caco-2

  14. Determinants for the activation and autoinhibition of the diguanylate cyclase response regulator WspR.

    PubMed

    De, Nabanita; Navarro, Marcos V A S; Raghavan, Rahul V; Sondermann, Holger

    2009-10-30

    The bacterial second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) controls secretion, cell adhesion, and motility, leading to biofilm formation and increased cytotoxicity. Diguanylate cyclases containing GGDEF and phosphodiesterases containing EAL or HD-GYP domains have been identified as the enzymes controlling cellular c-di-GMP levels, yet less is known regarding the molecular mechanisms governing regulation and signaling specificity. We recently determined a product-inhibition pathway for the diguanylate cyclase response regulator WspR from Pseudomonas, a potent molecular switch that controls biofilm formation. In WspR, catalytic activity is modulated by a helical stalk motif that connects its phospho-receiver and GGDEF domains. The stalks facilitate the formation of distinct oligomeric states that contribute to both activation and autoinhibition. Here, we provide novel insights into the regulation of diguanylate cyclase activity in WspR based on the crystal structures of full-length WspR, the isolated GGDEF domain, and an artificially dimerized catalytic domain. The structures highlight that inhibition is achieved by restricting the mobility of rigid GGDEF domains, mediated by c-di-GMP binding to an inhibitory site at the GGDEF domain. Kinetic measurements and biochemical characterization corroborate a model in which the activation of WspR requires the formation of a tetrameric species. Tetramerization occurs spontaneously at high protein concentration or upon addition of the phosphomimetic compound beryllium fluoride. Our analyses elucidate common and WspR-specific mechanisms for the fine-tuning of diguanylate cyclase activity.

  15. A Diguanylate Cyclase Acts as a Cell Division Inhibitor in a Two-Step Response to Reductive and Envelope Stresses

    PubMed Central

    Kim, Hyo Kyung

    2016-01-01

    ABSTRACT Cell division arrest is a universal checkpoint in response to environmental assaults that generate cellular stress. In bacteria, the cyclic di-GMP (c-di-GMP) signaling network is one of several signal transduction systems that regulate key processes in response to extra-/intracellular stimuli. Here, we find that the diguanylate cyclase YfiN acts as a bifunctional protein that produces c-di-GMP in response to reductive stress and then dynamically relocates to the division site to arrest cell division in response to envelope stress in Escherichia coli. YfiN localizes to the Z ring by interacting with early division proteins and stalls cell division by preventing the initiation of septal peptidoglycan synthesis. These studies reveal a new role for a diguanylate cyclase in responding to environmental change, as well as a novel mechanism for arresting cell division. PMID:27507823

  16. 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

  17. 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.

  18. Soluble guanylate cyclase modulators blunt hyperoxia effects on calcium responses of developing human airway smooth muscle.

    PubMed

    Britt, Rodney D; Thompson, Michael A; Kuipers, Ine; Stewart, Alecia; Vogel, Elizabeth R; Thu, James; Martin, Richard J; Pabelick, Christina M; Prakash, Y S

    2015-09-15

    Exposure to moderate hyperoxia in prematurity contributes to subsequent airway dysfunction and increases the risk of developing recurrent wheeze and asthma. The nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic GMP (cGMP) axis modulates airway tone by regulating airway smooth muscle (ASM) intracellular Ca(2+) ([Ca(2+)]i) and contractility. However, the effects of hyperoxia on this axis in the context of Ca(2+)/contractility are not known. In developing human ASM, we explored the effects of novel drugs that activate sGC independent of NO on alleviating hyperoxia (50% oxygen)-induced enhancement of Ca(2+) responses to bronchoconstrictor agonists. Treatment with BAY 41-2272 (sGC stimulator) and BAY 60-2770 (sGC activator) increased cGMP levels during exposure to 50% O2. Although 50% O2 did not alter sGCα1 or sGCβ1 expression, BAY 60-2770 did increase sGCβ1 expression. BAY 41-2272 and BAY 60-2770 blunted Ca(2+) responses to histamine in cells exposed to 50% O2. The effects of BAY 41-2272 and BAY 60-2770 were reversed by protein kinase G inhibition. These novel data demonstrate that BAY 41-2272 and BAY 60-2770 stimulate production of cGMP and blunt hyperoxia-induced increases in Ca(2+) responses in developing ASM. Accordingly, sGC stimulators/activators may be a useful therapeutic strategy in improving bronchodilation in preterm infants.

  19. Stimulation of hippocampal adenylyl cyclase activity dissociates memory consolidation processes for response and place learning.

    PubMed

    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 examined in mice given post-training intrahippocampal injections of forskolin (FK) aiming at stimulating hippocampal adenylyl cyclases (ACs). The injection was given at different time points over a period of 9 h following acquisition in either an appetitive bar-pressing task or water-maze tasks challenging respectively "response memory" and "place memory." Retention testing (24 h) showed that FK injection altered memory formation only when given within a 3- to 6-h time window after acquisition but yielded opposite memory effects as a function of task demands. Retention of the spatial task was impaired, whereas retention of both the cued-response in the water maze and the rewarded bar-press response were improved. Intrahippocampal injections of FK produced an increase in pCREB immunoreactivity, which was strictly limited to the hippocampus and lasted less than 2 h, suggesting that early effects (0-2 h) of FK-induced cAMP/CREB activation can be distinguished from late effects (3-6 h). These results delineate a consolidation period during which specific cAMP levels in the hippocampus play a crucial role in enhancing memory processes mediated by other brain regions (e.g., dorsal or ventral striatum) while eliminating interference by the formation of hippocampus-dependent memory.

  20. The prelimbic cortex muscarinic M₃ receptor-nitric oxide-guanylyl cyclase pathway modulates cardiovascular responses in rats.

    PubMed

    Fassini, Aline; Antero, Leandro S; Corrêa, Fernando M A; Joca, Sâmia R; Resstel, Leonardo B M

    2015-05-01

    The prelimbic cortex (PL), a limbic structure, sends projections to areas involved in the control of cardiovascular responses. Stimulation of the PL with acetylcholine (ACh) evokes depressor and tachycardiac responses mediated by local PL muscarinic receptors. Early studies demonstrated that stimulation of muscarinic receptors induced nitric oxide (NO) synthesis and cyclic guanosine cyclic monophosphate (cGMP) formation. Hence, this study investigates which PL muscarinic receptor subtype is involved in the cardiovascular response induced by ACh and tests the hypothesis that cardiovascular responses caused by muscarinic receptor stimulation in the PL are mediated by local NO and cGMP formation. PL pretreatment with J104129 (an M3 receptor antagonist) blocked the depressor and tachycardiac response evoked by injection of ACh into the PL. Pretreatment with either pirenzepine (an M1 receptor antagonist) or AF-DX 116 (an M2 and M4 receptor antagonist) did not affect cardiovascular responses evoked by ACh. Moreover, similarly to the antagonism of PL M3 receptors, pretreatment with N(ω)-propyl-L-arginine (an inhibitor of neuronal NO synthase), carboxy-PTIO(S)-3-carboxy-4-hydroxyphenylglicine (an NO scavenger), or 1H-[1,2,4]oxadiazolol-[4,3-a]quinoxalin-1-one (a guanylate cyclase inhibitor) blocked both the depressor and the tachycardiac response evoked by ACh. The current results demonstrate that cardiovascular responses evoked by microinjection of ACh into the PL are mediated by local activation of the M3 receptor-NO-guanylate cyclase pathway.

  1. Identification of a Hippeastrum hybridum guanylyl cyclase responsive to wounding and pathogen infection.

    PubMed

    Świeżawska, Brygida; Jaworski, Krzysztof; Szewczuk, Piotr; Pawełek, Agnieszka; Szmidt-Jaworska, Adriana

    2015-09-15

    Guanosine 3',5'-cyclic monophosphate (cGMP) is a critical component of many (patho)physiological processes in plants whilst guanylyl cyclases (GCs) which catalyse the formation of cGMP from GTP have remained somewhat elusive. Consequently, the two major aims are the discovery of novel guanylyl cyclases and the identification of GC/cGMP mediated processes. To identify a novel GC from Hippeastrum hybridum plant and facilitate the preparation of guanylyl cyclase in an amount sufficient for further crystallographic studies, we have constructed an overproduction system for this enzyme. This gene encodes a protein of 256 amino acids, with a calculated molecular mass of 28kD. The predicted amino acid sequence contains all the typical features and shows a high identity to other plant GCs. The GST-HpGC1 was catalytically active in Escherichia coli cells and the purified, recombinant HpGC1 was able to convert GTP to cGMP in the presence of divalent cations. The used overexpression system yields a guanylyl cyclase as 6% of the bacterial cytosolic protein. Besides the identification of HpGC1 as a guanylyl cyclase, the study has shown that the level of HpCG1 mRNA changed during stress conditions. Both mechanical damage and a Peyronellaea curtisii (=Phoma narcissi) fungi infection led to an initial decrease in the HpGC1 transcript level, followed by a substantial increase during the remainder of the 48-h test cycle. Moreover, significant changes in cyclic GMP level were observed, taking the form of oscillations. In conclusion, our data unequivocally identified the product of the HpGC1 gene as a guanylyl cyclase and demonstrates that such an overproduction system can be successfully used in enzyme synthesis. Furthermore, they indicate a link between the causing stimulus (wounding, infection) and guanylyl cyclase expression and the increase in cGMP amplitude. Therefore, it is concluded that appearance of cyclic GMP as a mediator in defense and wound-healing mechanisms provides a

  2. Cyclic AMP affects the haemocyte responses of larval Galleria mellonella to selected antigens.

    PubMed

    Marin, David; Dunphy, Gary B; Mandato, Craig A

    2005-05-01

    Signal transduction of the innate immediate responses of insect haemocytes to foreign matter is rarely considered. Herein using a combination of adenylate cyclase inhibitors and activators and phosphodiesterase inhibitors we determined that cyclic adenosine monophosphate (cAMP) at high levels normally impairs non-self response. Haemocyte contact with glass and bacteria lowered cAMP in vitro. Inactive phosphodiesterases, including type 4, impaired haemocyte reactions in vitro. Using the drugs in vivo to modulate adenylate cyclase and phosphodiesterases altered the total and types of haemocytes. Adenylate cyclase inhibitors and etazolate (a type 4 phosphodiesterase inhibitor) alone produced changes in the haemograms similar to those caused by Bacillus subtilis. Sequential injections of an enzyme modulator followed by B. subtilis impaired bacterial removal due (1) in the case of enzyme inhibitors, to the removal of haemocytes prior to bacterial challenge and (2) in the case of forskolin and IBMX to the shut-down of the haemocytes. Activating adenylate cyclase or inhibiting phosphodiesterase impaired bacterial removal when co-injecting the compounds and bacteria.

  3. Regulation of oxidative response and extracellular polysaccharide synthesis by a diadenylate cyclase in Streptococcus mutans.

    PubMed

    Cheng, Xingqun; Zheng, Xin; Zhou, Xuedong; Zeng, Jumei; Ren, Zhi; Xu, Xin; Cheng, Lei; Li, Mingyun; Li, Jiyao; Li, Yuqing

    2016-03-01

    Cyclic diadenosine monophosphate (c-di-AMP) has been implicated in the control of many important bacterial activities. However, the function of this molecule in Streptococcus mutans, the primary aetiological agent of human dental caries, is unknown. In this study, we identified and characterized a diadenylate cyclase, named CdaA, in S. mutans. Furthermore, we showed that in-frame deletion of the cdaA gene in S. mutans causes decreased c-di-AMP levels, increased sensitivity to hydrogen peroxide and increased production of extracellular polysaccharides. Global gene expression profiling revealed that more than 200 genes were significantly upregulated or downregulated (> 2.0-fold) in the cdaA mutant. Interestingly, genes with increased or decreased expression were clustered in cellular polysaccharide biosynthetic processes and oxidoreductase activity respectively. Notably, the expression of several genomic islands, such as GTF-B/C, TnSmu, CRISPR1-Cas and CRISPR2-Cas, was found to be altered in the cdaA mutant, indicating a possible link between these genomic islands and c-di-AMP signalling. Collectively, the results reported here show that CdaA is an important global modulator in S. mutans and is required for optimal growth and environmental adaption. This report also paves the way to unveil further the roles of c-di-AMP signalling networks in the biology and pathogenicity of S. mutans.

  4. 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

  5. Characterisation of two oxidosqualene cyclases responsible for triterpenoid biosynthesis in Ilex asprella.

    PubMed

    Zheng, Xiasheng; Luo, Xiuxiu; Ye, Guobing; Chen, Ye; Ji, Xiaoyu; Wen, Lingling; Xu, Yaping; Xu, Hui; Zhan, Ruoting; Chen, Weiwen

    2015-02-05

    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.

  6. Deficits in behavioral sensitization and dopaminergic responses to methamphetamine in adenylyl cyclase 1/8-deficient mice.

    PubMed

    Bosse, Kelly E; Charlton, Jennifer L; Susick, Laura L; Newman, Brooke; Eagle, Andrew L; Mathews, Tiffany A; Perrine, Shane A; Conti, Alana C

    2015-12-01

    The cAMP/protein kinase A pathway regulates methamphetamine (METH)-induced neuroplasticity underlying behavioral sensitization. We hypothesize that adenylyl cyclases (AC) 1/8 mediate these neuroplastic events and associated striatal dopamine regulation. Locomotor responses to METH (1 and 5 mg/kg) and striatal dopamine function were evaluated in mice lacking AC 1/8 (DKO) and wild-type (WT) mice. Only 5 mg/kg METH induced an acute locomotor response in DKO mice, which was significantly attenuated versus WT controls. DKO mice showed a marked attenuation in the development and expression of METH-induced behavioral sensitization across doses relative to WT controls. While basal and acute METH (5 mg/kg)-evoked accumbal dialysate dopamine levels were similar between genotypes, saline-treated DKO mice showed elevated tissue content of dopamine and homovanillic acid in the dorsal striatum (DS), reflecting dysregulated dopamine homeostasis and/or metabolism. Significant reductions in DS dopamine levels were observed in METH-sensitized DKO mice compared to saline-treated controls, an effect not observed in WT mice. Notably, saline-treated DKO mice had significantly increased phosphorylated Dopamine- and cAMP-regulated phosphoprotein levels, which were not further augmented following METH sensitization, as observed in WT mice. These data indicate that AC 1/8 are critical to mechanisms subserving drug-induced behavioral sensitization and mediate nigrostriatal pathway METH sensitivity. Calcium/calmodulin-stimulated adenylyl cyclase (AC) isoforms 1 and 8 were studied for their involvement in the adaptive neurobehavioral responses to methamphetamine. AC 1/8 double knockout (DKO) mice showed heightened basal locomotor activity and dorsal striatal dopamine responsivity. Conversely, methamphetamine-induced locomotor activity was attenuated in DKO mice, accompanied by reductions in dopamine and HVA content and impaired DARPP-32 activation. These findings indicate AC 1/8 signaling

  7. Challenge of human Jurkat T-cells with the adenylate cyclase activator forskolin elicits major changes in cAMP phosphodiesterase (PDE) expression by up-regulating PDE3 and inducing PDE4D1 and PDE4D2 splice variants as well as down-regulating a novel PDE4A splice variant.

    PubMed Central

    Erdogan, S; Houslay, M D

    1997-01-01

    The cAMP phosphodiesterase (PDE) 3 and PDE4 isoforms provide the major cAMP-hydrolysing PDE activities in Jurkat T-cells, with additional contributions from the PDE1 and PDE2 isoforms. Challenge of cells with the adenylate cyclase activator forskolin led to a rapid, albeit transient, increase in PDE3 activity occurring over the first 45 min, followed by a sustained increase in PDE3 activity which began after approximately 3 h and continued for at least 24 h. Only this second phase of increase in PDE3 activity was blocked by the transcriptional inhibitor actinomycin D. After approximately 3 h of exposure to forskolin, PDE4 activity had increased, via a process that could be inhibited by actinomycin D, and it remained elevated for at least a 24 h period. Such actions of forskolin were mimicked by cholera toxin and 8-bromo-cAMP. Forskolin increased intracellular cAMP concentrations in a time-dependent fashion and its action was enhanced when PDE induction was blocked with actinomycin D. Reverse transcription (RT)-PCR analysis, using generic primers designed to detect transcripts representing enzymically active products of the four PDE4 genes, identified transcripts for PDE4A and PDE4D but not for PDE4B or PDE4C in untreated Jurkat T-cells. Forskolin treatment did not induce transcripts for either PDE4B or PDE4C; however, it reduced the RT-PCR signal for PDE4A transcripts and markedly enhanced that for PDE4D transcripts. Using RT-PCR primers for PDE4 splice variants, a weak signal for PDE4D1 was evident in control cells whereas, in forskolin-treated cells, clear signals for both PDE4D1 and PDE4D2 were detected. RT-PCR analysis of the PDE4A species indicated that it was not the PDE4A isoform PDE-46 (PDE4A4B). Immunoblotting of control cells for PDE4 forms identified a single PDE4A species of approximately 118 kDa, which migrated distinctly from the PDE4A4B isoform PDE-46, with immunoprecipitation analyses showing that it provided all of the PDE4 activity in control

  8. Effect of gonadectomy of biochemical indices of striatal dopamine D/sub 1/ and D/sub 2/ receptors, their activity and adaptive response to antipsychotic drug treatment in rat

    SciTech Connect

    Jastrow, T.L.R.

    1987-01-01

    Endogenous gonadal steroids in male and female rats were removed by gonadectomy (Gnx). Striatal D/sub 1/ receptors and their activity were characterized by (/sup 3/H)SCH23390 binding parameters and D/sub 1/ receptor-stimulated adenylate cyclase activity in striatal membranes. Striatal D/sub 2/ receptors and their activity were characterized by (/sup 3/H)sulpiride binding parameters in striatal slices and D/sub 2/ receptor-mediated inhibition of forskolin-activated adenylate cyclase activity in striatal membranes. Sub-chronic D/sub 2/ receptor blockade consisted of the administration of the D/sub 2/ specific antagonist sulpiride (20 mg/kg) or vehicle i.p., 2x daily for 21 days followed by a 3 day drug withdrawal period. Gnx of female rats had no affect on striatal D/sub 1/-stimulated adenylate cyclase activity nor on the striatal D/sub 2/ receptor-mediated inhibition of forskolin-activated adenylate cyclase activity. Sub-chronic sulpiride treatment produced no adaptive changes in D/sub 1/-stimulated adenylate cyclase activity in sham-operated or Gnx female rats. Gnx of male rats produced a statistically significant 10% decrease in striatal (/sup 3/H)SCH23390 binding sites with no change in D/sub 1/-stimulated adenylate cyclase activity and no change in striatal(/sup 3/H)sulpiride binding parameters. Sub-chronic sulpiride treatment of sham-operated male rats produced a desensitization of the striatal D/sub 1/-stimulated adenylate cyclase activity with no change in the number of (/sup 3/H)SCH23390 binding sites and no change in (/sup 3/H)sulpiride binding parameters. Gnx of male rats blocked the development of the striatal D/sub 1/ receptor desensitization response elicited by sub-chronic sulpiride treatment, without affecting striatal (/sup 3/H)SCH23390 or (/sup 3/H)sulpiride binding parameters. We have demonstrated that sub-chronic D/sub 2/ receptor blockade in sham-operated male rats results in the desensitization of striatal D/sub 1/ receptor activity.

  9. Parasympathetic denervation increases responses to VIP in isolated rat parotid acini

    SciTech Connect

    McMillian, M.K.; Talamo, B.R. )

    1989-07-01

    Vasoactive intestinal peptide (VIP) is a putative neurotransmitter found in the salivary glands of many species, including the rat parotid gland. Parasympathetic denervation has been reported to deplete VIP in the rat parotid gland and to lead to supersensitivity to this peptide in vivo. We have compared the effects of VIP on acini isolated from parasympathetically denervated and unoperated parotid glands to examine possible supersensitivity to the peptide in vitro. VIP normally produced responses similar to those obtained with a low concentration of the beta adrenergic agonist isoproterenol (ISO), but strikingly different from the effects obtained with the muscarinic agonist carbachol (CARB). In parotid membrane preparations, VIP stimulated adenylate cyclase activity. Dissociated acini treated with VIP showed increases in cAMP accumulation and amylase release which were potentiated by forskolin and also by inhibition of phosphodiesterase. After parasympathetic denervation, maximal effects of VIP on adenylate cyclase, cAMP accumulation and amylase release in intact cells were increased two- to five-fold over contralateral control (or unoperated) parotid responses. The increase in adenylate cyclase-mediated responses after denervation was specific to VIP; there was no increased response nor increased sensitivity of any of these responses to ISO. Specific (125I)VIP binding to parotid acini increased two-fold per gland and three-fold per mg of protein after denervation; this probably explains the observed increases in the response to VIP.

  10. 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

    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.

  11. 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

  12. The Cyclase-associated protein Cap1 is important for proper regulation of infection-related morphogenesis in Magnaporthe oryzae.

    PubMed

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

    2012-09-01

    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

  13. Innate hemocyte responses of Malacosoma disstria larvae (C. Insecta) to antigens are modulated by intracellular cyclic AMP.

    PubMed

    Gulii, Vladislav; Dunphy, Gary B; Mandato, Craig A

    2009-08-01

    Invertebrate intracellular hemocyte signaling pathways affecting cellular-antigen responses, although defined for molluscs and some arthropods including dipteran insects, is less known for lepidopterans. Hemocytic-antigen responses of the arboreal pest lepidopteran Malacosoma disstria are linked to cAMP-dependent protein kinase A implicating cAMP in cellular hemocyte immune responses. The purpose in the present study was to determine intracellular cAMP effects on larval M. disstria hemocytes adhering to slides and bacteria. Altering adenylate cyclase and phosphodiesterase activities as well as cAMP levels in vitro and in vivo changed hemocyte responses to antigens. Quiescent hemocytes had high cAMP levels due to adenylate cyclase activity and possibly low phosphodiesterase (type 4) activity. Antigen contact diminished hemocytic cAMP levels. Inhibiting adenylate cyclase increased hemocyte-antigen and hemocyte-hemocyte adhesion, the latter producing nodules in vivo without bacterial antigens. Inhibiting phosphodiesterase type 4 produced the reverse effects. Pharmacologically increasing intracellular cAMP in attached hemocytes caused many of the cells to detach. Diminished intracellular cAMP changed hemograms in vivo in bacteria-free larvae comparable to changes induced by the bacterium, Bacillus subtilis, by producing nodules. Lowering cAMP enhanced also the removal of Xenorhabdus nematophila and B. subtilisin vivo.

  14. Effect of association with adenylyl cyclase-associated protein on the interaction of yeast adenylyl cyclase with Ras protein.

    PubMed

    Shima, F; Yamawaki-Kataoka, Y; Yanagihara, C; Tamada, M; Okada, T; Kariya, K; Kataoka, T

    1997-03-01

    Posttranslational modification of Ras protein has been shown to be critical for interaction with its effector molecules, including Saccharomyces cerevisiae adenylyl cyclase. However, the mechanism of its action was unknown. In this study, we used a reconstituted system with purified adenylyl cyclase and Ras proteins carrying various degrees of the modification to show that the posttranslational modification, especially the farnesylation step, is responsible for 5- to 10-fold increase in Ras-dependent activation of adenylyl cyclase activity even though it has no significant effect on their binding affinity. The stimulatory effect of farnesylation is found to depend on the association of adenylyl cyclase with 70-kDa adenylyl cyclase-associated protein (CAP), which was known to be required for proper in vivo response of adenylyl cyclase to Ras protein, by comparing the levels of Ras-dependent activation of purified adenylyl cyclase with and without bound CAP. The region of CAP required for this effect is mapped to its N-terminal segment of 168 amino acid residues, which coincides with the region required for the in vivo effect. Furthermore, the stimulatory effect is successfully reconstituted by in vitro association of CAP with the purified adenylyl cyclase molecule lacking the bound CAP. These results indicate that the association of adenylyl cyclase with CAP is responsible for the stimulatory effect of posttranslational modification of Ras on its activity and that this may be the mechanism underlying its requirement for the proper in vivo cyclic AMP response.

  15. Stress-related disorders, pituitary adenylate cyclase—activating peptide (PACAP)ergic system, and sex differences

    PubMed Central

    Ramikie, Teniel S.; Ressler, Kerry J.

    2016-01-01

    Trauma-related disorders, such as posttraumatic stress disorder (PTSD) are remarkably common and debilitating, and are often characterized by dysregulated threat responses. Across numerous epidemiological studies, females have been found to have an approximately twofold increased risk for PTSD and other stress-related disorders. Understanding the biological mechanisms of this differential risk is of critical importance. Recent data suggest that the pituitary adenylate cyclase-activating polypeptide (PACAP) pathway is a critical regulator of the stress response across species. Moreover, increasing evidence suggests that this pathway is regulated by both stress and estrogen modulation and may provide an important window into understanding mechanisms of sex differences in the stress response. We have recently shown that PACAP and its receptor (PAC1R) are critical mediators of abnormal processes after psychological trauma. Notably, in heavily traumatized human subjects, there appears to be a robust sex-specific association of PACAP blood levels and PAC1R gene variants with fear physiology, PTSD diagnosis, and symptoms, specifically in females. The sex-specific association occurs within a single-nucleotide polymorphism (rs2267735) that resides in a putative estrogen response element involved in PAC1R gene regulation. Complementing these human data, the PAC1R messenger RNA is induced with fear conditioning or estrogen replacement in rodent models. These data suggest that perturbations in the PACAP-PAC1R pathway are regulated by estrogen and are involved in abnormal fear responses underlying PTSD. PMID:28179812

  16. Role of the bicarbonate-responsive soluble adenylyl cyclase in pH sensing and metabolic regulation

    PubMed Central

    Chang, Jung-Chin; Oude-Elferink, Ronald P. J.

    2014-01-01

    The evolutionarily conserved soluble adenylyl cyclase (sAC, adcy10) was recently identified as a unique source of cAMP in the cytoplasm and the nucleus. Its activity is regulated by bicarbonate and fine-tuned by calcium. As such, and in conjunction with carbonic anhydrase (CA), sAC constitutes an HCO−3/CO−2/pH sensor. In both alpha-intercalated cells of the collecting duct and the clear cells of the epididymis, sAC is expressed at significant level and involved in pH homeostasis via apical recruitment of vacuolar H+-ATPase (VHA) in a PKA-dependent manner. In addition to maintenance of pH homeostasis, sAC is also involved in metabolic regulation such as coupling of Krebs cycle to oxidative phosphorylation via bicarbonate/CO2 sensing. Additionally, sAC also regulates CFTR channel and plays an important role in regulation of barrier function and apoptosis. These observations suggest that sAC, via bicarbonate-sensing, plays an important role in maintaining homeostatic status of cells against fluctuations in their microenvironment. PMID:24575049

  17. Adenylating Enzymes in Mycobacterium tuberculosis as Drug Targets

    PubMed Central

    Duckworth, Benjamin P.; Nelson, Kathryn M.; Aldrich, Courtney C.

    2013-01-01

    Adenylation or adenylate-forming enzymes (AEs) are widely found in nature and are responsible for the activation of carboxylic acids to intermediate acyladenylates, which are mixed anhydrides of AMP. In a second reaction, AEs catalyze the transfer of the acyl group of the acyladenylate onto a nucleophilic amino, alcohol, or thiol group of an acceptor molecule leading to amide, ester, and thioester products, respectively. Mycobacterium tuberculosis encodes for more than 60 adenylating enzymes, many of which represent potential drug targets due to their confirmed essentiality or requirement for virulence. Several strategies have been used to develop potent and selective AE inhibitors including high-throughput screening, fragment-based screening, and the rationale design of bisubstrate inhibitors that mimic the acyladenylate. In this review, a comprehensive analysis of the mycobacterial adenylating enzymes will be presented with a focus on the identification of small molecule inhibitors. Specifically, this review will cover the aminoacyl tRNA-synthetases (aaRSs), MenE required for menaquinone synthesis, the FadD family of enzymes including the fatty acyl-AMP ligases (FAAL) and the fatty acyl-CoA ligases (FACLs) involved in lipid metabolism, and the nonribosomal peptide synthetase adenylation enzyme MbtA that is necessary for mycobactin synthesis. Additionally, the enzymes NadE, GuaA, PanC, and MshC involved in the respective synthesis of NAD, guanine, pantothenate, and mycothiol will be discussed as well as BirA that is responsible for biotinylation of the acyl CoA-carboxylases. PMID:22283817

  18. Regulation of Neuronal Oxygen Responses in C. elegans Is Mediated through Interactions between Globin 5 and the H-NOX Domains of Soluble Guanylate Cyclases

    PubMed Central

    Abergel, Zohar; Chatterjee, Arijit Kumar; Zuckerman, Binyamin

    2016-01-01

    Soluble guanylate cyclases (sGCs) are gas-binding proteins that control diverse physiological processes such as vasodilation, platelet aggregation, and synaptic plasticity. In the nematode Caenorhabditis elegans, a complex of sGCs, GCY-35 and GCY-36, functions in oxygen (O2) sensing. Previous studies suggested that the neuroglobin GLB-5 genetically interacts with GCY-35, and that the inhibitory effect of GLB-5 on GCY-35 function is necessary for fast recovery from prolonged hypoxia. In this study, we identified mutations in gcy-35 and gcy-36 that impact fast recovery and other phenotypes associated with GLB-5, without undermining sGC activity. These mutations, heb1 and heb3, change conserved amino acid residues in the regulatory H-NOX domains of GCY-35 and GCY-36, respectively, and appear to suppress GLB-5 activity by different mechanisms. Moreover, we observed that short exposure to 35% O2 desensitized the neurons responsible for ambient O2 sensing and that this phenomenon does not occur in heb1 animals. These observations may implicate sGCs in neuronal desensitization mechanisms far beyond the specific case of O2 sensing in nematodes. The conservation of functionally important regions of sGCs is supported by examining site-directed mutants of GCY-35, which suggested that similar regions in the H-NOX domains of O2 and NO-sensing sGCs are important for heme/gas interactions. Overall, our studies provide novel insights into sGC activity and regulation, and implicate similar structural determinants in the control of both O2 and NO sensors. SIGNIFICANCE STATEMENT Soluble guanylate cyclases (sGCs) control essential and diverse physiological processes, including memory processing. We used Caenorhabditis elegans to explore how a neuroglobin inhibits a complex of oxygen-sensing sGCs, identifying sGC mutants that resist inhibition. Resistance appears to arise by two different mechanisms: increased basal sGC activity or disruption of an interaction with neuroglobin. Our

  19. Marine Bivalve Cellular Responses to Beta Blocker Exposures

    EPA Science Inventory

    β blockers are prescription drugs used for medical treatment of hypertension and arrhythmias. They prevent binding of agonists such as catecholamines to β adrenoceptors. In the absence of agonist induced activation of the receptor, adenylate cyclase is not activated whi...

  20. Activation of IKK/NF-κB provokes renal inflammatory responses in guanylyl cyclase/natriuretic peptide receptor-A gene-knockout mice

    PubMed Central

    Das, Subhankar; Periyasamy, Ramu

    2012-01-01

    The present study was aimed at determining the consequences of the disruption of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene (Npr1) on proinflammatory responses of nuclear factor kappa B, inhibitory kappa B kinase, and inhibitory kappa B alpha (NF-κB, IKK, IκBα) in the kidneys of mutant mice. The results showed that the disruption of Npr1 enhanced the renal NF-κB binding activity by 3.8-fold in 0-copy (−/−) mice compared with 2-copy (+/+) mice. In parallel, IKK activity and IκBα protein phosphorylation were increased by 8- and 11-fold, respectively, in the kidneys of 0-copy mice compared with wild-type mice. Interestingly, IκBα was reduced by 80% and the expression of proinflammatory cytokines and renal fibrosis were significantly enhanced in 0-copy mice than 2-copy mice. Treatment of 0-copy mice with NF-κB inhibitors andrographolide, pyrrolidine dithiocarbamate, and etanercept showed a substantial reduction in renal fibrosis, attenuation of proinflammatory cytokines gene expression, and significantly reduced IKK activity and IkBα phosphorylation. These findings indicate that the systemic disruption of Npr1 activates the renal NF-κB pathways in 0-copy mice, which transactivates the expression of various proinflammatory cytokines to initiate renal remodeling; however, inhibition of NF-κB pathway repairs the abnormal renal pathology in mutant mice. PMID:22318993

  1. Actin activates Pseudomonas aeruginosa ExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxins

    PubMed Central

    Belyy, Alexander; Raoux-Barbot, Dorothée; Saveanu, Cosmin; Namane, Abdelkader; Ogryzko, Vasily; Worpenberg, Lina; David, Violaine; Henriot, Veronique; Fellous, Souad; Merrifield, Christien; Assayag, Elodie; Ladant, Daniel; Renault, Louis; Mechold, Undine

    2016-01-01

    The nucleotidyl cyclase toxin ExoY is one of the virulence factors injected by the Pseudomonas aeruginosa type III secretion system into host cells. Inside cells, it is activated by an unknown eukaryotic cofactor to synthesize various cyclic nucleotide monophosphates. ExoY-like adenylate cyclases are also found in Multifunctional-Autoprocessing Repeats-in-ToXin (MARTX) toxins produced by various Gram-negative pathogens. Here we demonstrate that filamentous actin (F-actin) is the hitherto unknown cofactor of ExoY. Association with F-actin stimulates ExoY activity more than 10,000 fold in vitro and results in stabilization of actin filaments. ExoY is recruited to actin filaments in transfected cells and alters F-actin turnover. Actin also activates an ExoY-like adenylate cyclase MARTX effector domain from Vibrio nigripulchritudo. Finally, using a yeast genetic screen, we identify actin mutants that no longer activate ExoY. Our results thus reveal a new sub-group within the class II adenylyl cyclase family, namely actin-activated nucleotidyl cyclase (AA-NC) toxins. PMID:27917880

  2. A cyanobacterial light activated adenylyl cyclase partially restores development of a Dictyostelium discoideum, adenylyl cyclase a null mutant.

    PubMed

    Chen, Zhi-Hui; Raffelberg, Sarah; Losi, Aba; Schaap, Pauline; Gärtner, Wolfgang

    2014-12-10

    A light-regulated adenylyl cyclase, mPAC, was previously identified from the cyanobacterium Microcoleus chthonoplastes PCC7420. MPAC consists of a flavin-based blue light-sensing LOV domain and a catalytic domain. In this work, we expressed mPAC in an adenylate cyclase A null mutant (aca-) of the eukaryote Dictyostelium discoideum and tested to what extent light activation of mPAC could restore the cAMP-dependent developmental programme of this organism. Amoebas of Dictyostelium, a well-established model organism, generate and respond to cAMP pulses, which cause them to aggregate and construct fruiting bodies. mPAC was expressed under control of a constitutive actin-15 promoter in D. discoideum and displayed low basal adenylyl cyclase activity in darkness that was about five-fold stimulated by blue light. mPAC expression in aca- cells marginally restored aggregation and fruiting body formation in darkness. However, more and larger fruiting bodies were formed when mPAC expressing cells were incubated in light. Extending former applications of light-regulated AC, these results demonstrate that mPAC can be used to manipulate multicellular development in eukaryotes in a light dependent manner.

  3. Aprataxin resolves adenylated RNA–DNA junctions to maintain genome integrity

    SciTech Connect

    Tumbale, Percy; Williams, Jessica S.; Schellenberg, Matthew J.; Kunkel, Thomas A.; Williams, R. Scott

    2013-12-22

    Faithful maintenance and propagation of eukaryotic genomes is ensured by three-step DNA ligation reactions used by ATP-dependent DNA ligases. Paradoxically, when DNA ligases encounter nicked DNA structures with abnormal DNA termini, DNA ligase catalytic activity can generate and/or exacerbate DNA damage through abortive ligation that produces chemically adducted, toxic 5'-adenylated (5'-AMP) DNA lesions. Aprataxin (APTX) reverses DNA adenylation but the context for deadenylation repair is unclear. Here we examine the importance of APTX to RNase-H2-dependent excision repair (RER) of a lesion that is very frequently introduced into DNA, a ribonucleotide. We show that ligases generate adenylated 5' ends containing a ribose characteristic of RNase H2 incision. APTX efficiently repairs adenylated RNA–DNA, and acting in an RNA–DNA damage response (RDDR), promotes cellular survival and prevents S-phase checkpoint activation in budding yeast undergoing RER. Structure–function studies of human APTX–RNA–DNA–AMP–Zn complexes define a mechanism for detecting and reversing adenylation at RNA–DNA junctions. This involves A-form RNA binding, proper protein folding and conformational changes, all of which are affected by heritable APTX mutations in ataxia with oculomotor apraxia 1. Together, these results indicate that accumulation of adenylated RNA–DNA may contribute to neurological disease.

  4. Plant growth-promoting hormones activate mammalian guanylate cyclase activity.

    PubMed

    Vesely, D L; Hudson, J L; Pipkin, J L; Pack, L D; Meiners, S E

    1985-05-01

    In vivo injections of plant growth-promoting hormones increase the growth of animals as well as plants. Plant growth-promoting hormones and positive plant growth regulators are known to increase RNA and protein synthesis. Since cyclic GMP also increases RNA and protein synthesis, the object of the present investigation was to determine whether physiological levels of plant growth-promoting hormones and positive plant growth regulators have part of their mechanism(s) of action through stimulation of the guanylate cyclase (EC 4.6.1.2)-cyclic GMP system. Representatives of the three classes of growth-promoting hormones were investigated. Thus, auxins (indole-3-acetic acid, indole-3-butyric acid, beta-naphthoxyacetic acid, and 2,4,5-trichlorophenoxy acetic acid), gibberellins (gibberellic acid), and cytokinins [N6-benzyl adenine, kinetin (6-furfuryl aminopurine), and beta-(2-furyl) acrylic acid] all increased rat lung, small intestine, liver, and renal cortex guanylate cyclase activity 2- to 4-fold at the 1 microM concentration. Dose response curves revealed that maximal stimulation of guanylate cyclase by these plant growth regulators was at 1 microM; there was no augmented cyclase activity at 1 nM. The guanylate cyclase cationic cofactor manganese was not essential for augmentation of guanylate cyclase by these plant growth-promoting regulators. The antioxidant butylated hydroxytoluene did not block the enhancement of guanylate cyclase by these plant growth-promoting factors. These data suggest that guanylate cyclase may play a role in the mechanism of action of plant growth-promoting hormones and even of positive plant regulators at the cellular level.

  5. Altered adrenergic response and specificity of the receptors in rat ascites hepatoma AH130.

    PubMed

    Sanae, F; Miyamoto, K; Koshiura, R

    1989-11-15

    Adenylate cyclase activation through adrenergic receptors in rat ascites hepatoma (AH) 130 cells in response to adrenergic drugs was studied, and receptor binding and displacement were compared with those of normal rat hepatocytes. Epinephrine (Epi) and norepinephrine (NE) activated AH130 adenylate cyclase about half as much as isoproterenol (IPN) but equaled IPN after treatment with the alpha-antagonist phentolamine or islet-activating protein (IAP). The three catecholamines in hepatocytes were similar regardless of phentolamine or IAP. These catecholamines activated adenylate cyclase in order of IPN greater than NE greater than Epi in AH130 cells but IPN greater than Epi greater than NE in hepatocytes. We then used the alpha 1-selective ligand [3H]prazosin, the alpha 2-selective ligand [3H]clonidine, and the beta-ligand [125I]iodocyanopindolol [( 125I]ICYP), and found that AH130 cells had few prazosin-binding sites, about eight times as many clonidine-binding sites with high affinity, and many more ICYP-binding sites than in hepatocytes. The dissociation constant (Ki) of the beta 1-selective drug metoprolol by Hofstee plots for AH130 cells was lower than that for hepatocytes. The inhibition of specific ICYP binding by the beta 2-selective agonist salbutamol for AH130 cells gave only one Ki value which was much higher than both high and low Ki values of the drug for hepatocytes. These findings indicate that the alpha- and beta-adrenergic receptors in hepatocytes are predominantly alpha 1-type and beta 2-type, but that those in AH130 cells are predominantly alpha 2-type and beta 1-type, and the low adrenergic response of AH130 cells is due to the dominant appearance of alpha 2-adrenergic receptors, linked with the inhibitory guanine-nucleotide binding regulatory protein, instead of alpha 1-adrenergic receptors, and beta 1-adrenergic receptors with low affinity for the hormone.

  6. Myocardial adrenergic responsiveness after lethal and nonlethal doses of endotoxin

    SciTech Connect

    Shepherd, R.E.; Lang, C.H.; McDonough, K.H.

    1987-02-01

    A dose-dependent impairment of intrinsic myocardial performance has been observed following in vivo administration of endotoxin. The present study reports a dose-dependent increase in plasma catecholamines following endotoxin (ET) that may impair ..beta..-adrenergic responsiveness. Hearts were removed from pentobarbital-anesthetized rats 4 h after a bolus injection of saline or ET and were studied as isolated cell preparations following collagenase digestion. Responsiveness of isoproterenol-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in myocytes prepared from hearts of animals injected with 10 and 100 ..mu..g ET was decreased when compared with control rats and was significantly blunted in myocytes prepared from animals receiving 1000 ..mu..g ET. Similar sensitivities of the cAMP system existed, as judged by similar half-maximum effective concentration values. cAMP accumulation in the presence of 1 ..mu..M forskolin was depressed in myocytes from the 1000-..mu..g ET animals; ..beta..-adrenergic receptor density was decreased 25% in myocytes from high-dose ET animals when compared with control animals. This was accompanied by a nonsignificant reduction in the affinity of binding sites for (+/-)(/sup 3/H)CGP 12177. The blunted myocyte hormonal responsiveness following ET challenge appears to be related to the decreased activity of the adenylate cyclase that may be attributed to alterations in both receptor density and in the adenylate cyclase itself.

  7. The 70-kilodalton adenylyl cyclase-associated protein is not essential for interaction of Saccharomyces cerevisiae adenylyl cyclase with RAS proteins.

    PubMed

    Wang, J; Suzuki, N; Kataoka, T

    1992-11-01

    In the yeast Saccharomyces cerevisiae, adenylyl cyclase is regulated by RAS proteins. We show here that the yeast adenylyl cyclase forms at least two high-molecular-weight complexes, one with the RAS protein-dependent adenylyl cyclase activity and the other with the Mn(2+)-dependent activity, which are separable by their size difference. The 70-kDa adenylyl cyclase-associated protein (CAP) existed in the former complex but not in the latter. Missense mutations in conserved motifs of the leucine-rich repeats of the catalytic subunit of adenylyl cyclase abolished the RAS-dependent activity, which was accompanied by formation of a very high molecular weight complex having the Mn(2+)-dependent activity. Contrary to previous results, disruption of the gene encoding CAP did not alter the extent of RAS protein-dependent activation of adenylyl cyclase, while a concomitant decrease in the size of the RAS-responsive complex was observed. These results indicate that CAP is not essential for interaction of the yeast adenylyl cyclase with RAS proteins even though it is an inherent component of the RAS-responsive adenylyl cyclase complex.

  8. Adenylate Kinase and AMP Signaling Networks: Metabolic Monitoring, Signal Communication and Body Energy Sensing

    PubMed Central

    Dzeja, Petras; Terzic, Andre

    2009-01-01

    Adenylate kinase and downstream AMP signaling is an integrated metabolic monitoring system which reads the cellular energy state in order to tune and report signals to metabolic sensors. A network of adenylate kinase isoforms (AK1-AK7) are distributed throughout intracellular compartments, interstitial space and body fluids to regulate energetic and metabolic signaling circuits, securing efficient cell energy economy, signal communication and stress response. The dynamics of adenylate kinase-catalyzed phosphotransfer regulates multiple intracellular and extracellular energy-dependent and nucleotide signaling processes, including excitation-contraction coupling, hormone secretion, cell and ciliary motility, nuclear transport, energetics of cell cycle, DNA synthesis and repair, and developmental programming. Metabolomic analyses indicate that cellular, interstitial and blood AMP levels are potential metabolic signals associated with vital functions including body energy sensing, sleep, hibernation and food intake. Either low or excess AMP signaling has been linked to human disease such as diabetes, obesity and hypertrophic cardiomyopathy. Recent studies indicate that derangements in adenylate kinase-mediated energetic signaling due to mutations in AK1, AK2 or AK7 isoforms are associated with hemolytic anemia, reticular dysgenesis and ciliary dyskinesia. Moreover, hormonal, food and antidiabetic drug actions are frequently coupled to alterations of cellular AMP levels and associated signaling. Thus, by monitoring energy state and generating and distributing AMP metabolic signals adenylate kinase represents a unique hub within the cellular homeostatic network. PMID:19468337

  9. Coiled-coil interaction of N-terminal 36 residues of cyclase-associated protein with adenylyl cyclase is sufficient for its function in Saccharomyces cerevisiae ras pathway.

    PubMed

    Nishida, Y; Shima, F; Sen, H; Tanaka, Y; Yanagihara, C; Yamawaki-Kataoka, Y; Kariya, K; Kataoka, T

    1998-10-23

    In the budding yeast Saccharomyces cerevisiae, association with the 70-kDa cyclase-associated protein (CAP) is required for proper response of adenylyl cyclase to Ras proteins. We show here that a small segment comprising the N-terminal 36 amino acid residues of CAP is sufficient for association with adenylyl cyclase as well as for its function in the Ras-adenylyl cyclase pathway as assayed by the ability to confer RAS2(Val-19)-dependent heat shock sensitivity to yeast cells. The CAP-binding site of adenylyl cyclase was mapped to a segment of 119 amino acid residues near its C terminus. Both of these regions contained tandem repetitions of a heptad motif alphaXXalphaXXX (where alpha represents a hydrophobic amino acid and X represents any amino acid), suggesting a coiled-coil interaction. When mutants of CAP defective in associating with adenylyl cyclase were isolated by screening of a pool of randomly mutagenized CAP, they were found to carry substitution mutations in one of the key hydrophobic residues in the heptad repeats. Furthermore, mutations of the key hydrophobic residues in the heptad repeats of adenylyl cyclase also resulted in loss of association with CAP. These results indicate the coiled-coil mechanism as a basis of the CAP-adenylyl cyclase interaction.

  10. Identification of a D1 dopamine receptor, not linked to adenylate cyclase, on lactotroph cells.

    PubMed Central

    Schoors, D. F.; Vauquelin, G. P.; De Vos, H.; Smets, G.; Velkeniers, B.; Vanhaelst, L.; Dupont, A. G.

    1991-01-01

    1. We studied the lactotroph cells of the rat by both in vivo and in vitro pharmacological techniques for the presence of D1-receptors. Both approaches revealed the presence of D2-receptor, stimulated by quinpirole (resulting in an inhibition of prolactin secretion) and blocked by domperidone. 2. Administration of fenoldopam, the most selective D1-receptor agonist currently available, resulted in a dose-dependent decrease of prolactin secretion in vivo (after pretreatment with alpha-methyl-p-tyrosine) and in vitro (cultured pituitary cells). This increase was dose-dependently blocked by the selective D1-receptor antagonist, SCH 23390, and although the effect of fenoldopam was less than that obtained by D2-receptor stimulation, these data suggest that a D1-receptor also controls prolactin secretion. 3. In order to detect the location of these dopamine receptors, autoradiographic studies were performed by use of [3H]-SCH 23390 and [3H]-spiperone as markers for D1- and D2-receptors, respectively. Specific binding sites for [3H]-SCH 23390 were demonstrated. Fenoldopam dose-dependently reduced [3H]-SCH 23390 binding, but had no effect on [3H]-spiperone binding. Immunocytochemical labelling of prolactin cells after incubation with [3H]-SCH 23390 revealed that the granulae and hence, D1 binding sites were present on the lactotroph cells. 4. Radioligand binding studies performed on membranes from anterior pituitary cells revealed the presence of the D2-receptor (54 fmol mg-1 protein) with a Kd of 0.58 nM for [3H]-spiperone, but failed to detect D1-receptors. 5. Finally, we studied the effect of dopamine and of fenoldopam on the adenosine 3':5'-cyclic monophosphate (cyclic AMP) content of anterior pituitary cells.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 4 Figure 5 PMID:1833020

  11. Replacement of Adenylate Cyclase Toxin in a Prominent Lineage of Bordetella bronchiseptica

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bordetella bronchiseptica is a gram negative respiratory pathogen that infects a wide-range of hosts and causes a diverse spectrum of disease that may be due in part to the set of virulence factors utilized by different strains. In a murine model of infection, we found that virulence, as measured by...

  12. Role for Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) in Cystitis-induced Plasticity of Micturition Reflexes

    PubMed Central

    Braas, Karen M.; May, Victor; Zvara, Peter; Nausch, Bernhard; Kliment, Jan; Dunleavy, J. Dana; Nelson, Mark T.; Vizzard, Margaret A.

    2006-01-01

    PACAP peptides are expressed and regulated in sensory afferents of the micturition pathway. Although these studies have implicated PACAP in bladder control, the physiological significance of these observations has not been firmly established. To clarify these issues, the roles of PACAP and PACAP signaling in micturition and cystitis were examined in receptor characterization and physiological assays. PACAP receptors were identified in various tissues of the micturition pathway including bladder detrusor smooth muscle and urothelium. Bladder smooth muscle expressed heterogeneously PAC1null, PAC1HOP1 and VPAC2 receptors; the urothelium was more restricted in expressing preferentially the PAC1 receptor subtype only. Immunocytochemical studies for PAC1 receptors were consistent with these tissue distributions. Furthermore, the addition of 50 – 100 nM PACAP27 or PACAP38 to isolated bladder strips elicited transient contractions and sustained increases in the amplitude of spontaneous phasic contractions. Treatment of the bladder strips with tetrodotoxin (1 μM) did not alter the spontaneous phasic contractions suggesting direct PACAP effects on bladder smooth muscle. PACAP also increased the amplitude of nerve-evoked contractions. By contrast, VIP had no direct effects on bladder smooth muscle. In a rat cyclophosphamide (CYP)-induced cystitis paradigm, intrathecal or intravesical administration of PAC1 receptor antagonist, PACAP6-38, reduced cystitis-induced bladder overactivity. In sum, these studies support roles for PACAP in micturition and suggest that inflammation-induced plasticity in PACAP expression in peripheral and central micturition pathways contribute to bladder dysfunction with cystitis. PMID:16322346

  13. Analysis of the function of the 70-kilodalton cyclase-associated protein (CAP) by using mutants of yeast adenylyl cyclase defective in CAP binding.

    PubMed

    Wang, J; Suzuki, N; Nishida, Y; Kataoka, T

    1993-07-01

    In Saccharomyces cerevisiae, adenylyl cyclase forms a complex with the 70-kDa cyclase-associated protein (CAP). By in vitro mutagenesis, we assigned a CAP-binding site of adenylyl cyclase to a small segment near its C terminus and created mutants which lost the ability to bind CAP. CAP binding was assessed first by observing the ability of the overproduced C-terminal 150 residues of adenylyl cyclase to sequester CAP, thereby suppressing the heat shock sensitivity of yeast cells bearing the activated RAS2 gene (RAS2Val-19), and then by immunoprecipitability of adenylyl cyclase activity with anti-CAP antibody and by direct measurement of the amount of CAP bound. Yeast cells whose chromosomal adenylyl cyclase genes were replaced by the CAP-nonbinding mutants possessed adenylyl cyclase activity fully responsive to RAS2 protein in vitro. However, they did not exhibit sensitivity to heat shock in the RAS2Val-19 background. When glucose-induced accumulation of cyclic AMP (cAMP) was measured in these mutants carrying RAS2Val-19, a rapid transient rise indistinguishable from that of wild-type cells was observed and a high peak level and following persistent elevation of the cAMP concentration characteristic of RAS2Val-19 were abolished. In contrast, in the wild-type RAS2 background, similar cyclase gene replacement did not affect the glucose-induced cAMP response. These results suggest that the association with CAP, although not involved in the in vivo response to the wild-type RAS2 protein, is somehow required for the exaggerated response of adenylyl cyclase to activated RAS2.

  14. Adenylate kinase 1 knockout mice have normal thiamine triphosphate levels.

    PubMed

    Makarchikov, Alexander F; Wins, Pierre; Janssen, Edwin; Wieringa, Bé; Grisar, Thierry; Bettendorff, Lucien

    2002-10-21

    Thiamine triphosphate (ThTP) is found at low concentrations in most animal tissues and it may act as a phosphate donor for the phosphorylation of proteins, suggesting a potential role in cell signaling. Two mechanisms have been proposed for the enzymatic synthesis of ThTP. A thiamine diphosphate (ThDP) kinase (ThDP+ATP if ThTP+ADP) has been purified from brewer's yeast and shown to exist in rat liver. However, other data suggest that, at least in skeletal muscle, adenylate kinase 1 (AK1) is responsible for ThTP synthesis. In this study, we show that AK1 knockout mice have normal ThTP levels in skeletal muscle, heart, brain, liver and kidney, demonstrating that AK1 is not responsible for ThTP synthesis in those tissues. We predict that the high ThTP content of particular tissues like the Electrophorus electricus electric organ, or pig and chicken skeletal muscle is more tightly correlated with high ThDP kinase activity or low soluble ThTPase activity than with non-stringent substrate specificity and high activity of adenylate kinase.

  15. Marihuana-derived material: biochemical studies of the ocular responses.

    PubMed

    Green, K; Cheeks, K; Mittag, T; Riley, M V; Symonds, C M; Deutsch, H M; Hodges, L C; Zalkow, L H

    1985-05-01

    Some biochemical factors of the iris-ciliary body of the rabbit have been examined for effects induced by water-soluble marihuana-derived material (MDM). Adenylate cyclase activity and sensitivity to beta-adrenergic agonists were unchanged, as measured 4 hours after MDM administration in vivo. Magnesium-dependent and anion-sensitive, but not sodium-potassium, ATPase activities were inhibited 6 hours after MDM administration in vivo, although they were unaffected by in vitro incubation. Topical administration of a potent substance P antagonist had no effect on the time course or magnitude of intravenous MDM-induced ocular effects in rabbit. Intravenously administered sugars antagonized the effects of MDM on intraocular pressure. A variety of drugs which display a range of biochemical effects varying from beta-adrenergic receptor agonism, to alteration of glycoprotein residues were employed. None of the agents employed, ranging from cAMP modifiers to protein synthesis blockers, had any effect on the MDM-induced response. It is apparent that the mechanism underlying the ocular hypotensive effect of MDM does not reside in mediation through adenylate cyclase, ATPase or substance P, but rather through a mechanism mediated by terminal sugar moieties on the molecule. The data suggest that modification of the surface membrane glycoprotein residues on the ciliary epithelium can induce marked alterations in aqueous humor flow rate.

  16. Interleukin 1 and Tumor Necrosis Factor Inhibit Cardiac Myocyte β -adrenergic Responsiveness

    NASA Astrophysics Data System (ADS)

    Gulick, Tod; Chung, Mina K.; Pieper, Stephen J.; Lange, Louis G.; Schreiner, George F.

    1989-09-01

    Reversible congestive heart failure can accompany cardiac allograft rejection and inflammatory myocarditis, conditions associated with an immune cell infiltrate of the myocardium. To determine whether immune cell secretory products alter cardiac muscle metabolism without cytotoxicity, we cultured cardiac myocytes in the presence of culture supernatants from activated immune cells. We observed that these culture supernatants inhibit β -adrenergic agonist-mediated increases in cultured cardiac myocyte contractility and intracellular cAMP accumulation. The myocyte contractile response to increased extracellular Ca2+ concentration is unaltered by prior exposure to these culture supernatants, as is the increase in myocyte intracellular cAMP concentration in response to stimulation with forskolin, a direct adenyl cyclase activator. Inhibition occurs in the absence of alteration in β -adrenergic receptor density or ligand binding affinity. Suppressive activity is attributable to the macrophage-derived cytokines interleukin 1 and tumor necrosis factor. Thus, these observations describe a role for defined cytokines in regulating the hormonal responsiveness and function of contractile cells. The effects of interleukin 1 and tumor necrosis factor on intracellular cAMP accumulation may be a model for immune modulation of other cellular functions dependent upon cyclic nucleotide metabolism. The uncoupling of agonist-occupied receptors from adenyl cyclase suggests that β -receptor or guanine nucleotide binding protein function is altered by the direct or indirect action of cytokines on cardiac muscle cells.

  17. 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

  18. Characterization of the active site of ADP-ribosyl cyclase.

    PubMed

    Munshi, C; Thiel, D J; Mathews, I I; Aarhus, R; Walseth, T F; Lee, H C

    1999-10-22

    ADP-ribosyl cyclase synthesizes two Ca(2+) messengers by cyclizing NAD to produce cyclic ADP-ribose and exchanging nicotinic acid with the nicotinamide group of NADP to produce nicotinic acid adenine dinucleotide phosphate. Recombinant Aplysia cyclase was expressed in yeast and co-crystallized with a substrate, nicotinamide. x-ray crystallography showed that the nicotinamide was bound in a pocket formed in part by a conserved segment and was near the central cleft of the cyclase. Glu(98), Asn(107) and Trp(140) were within 3.5 A of the bound nicotinamide and appeared to coordinate it. Substituting Glu(98) with either Gln, Gly, Leu, or Asn reduced the cyclase activity by 16-222-fold, depending on the substitution. The mutant N107G exhibited only a 2-fold decrease in activity, while the activity of W140G was essentially eliminated. The base exchange activity of all mutants followed a similar pattern of reduction, suggesting that both reactions occur at the same active site. In addition to NAD, the wild-type cyclase also cyclizes nicotinamide guanine dinucleotide to cyclic GDP-ribose. All mutant enzymes had at least half of the GDP-ribosyl cyclase activity of the wild type, some even 2-3-fold higher, indicating that the three coordinating amino acids are responsible for positioning of the substrate but not absolutely critical for catalysis. To search for the catalytic residues, other amino acids in the binding pocket were mutagenized. E179G was totally devoid of GDP-ribosyl cyclase activity, and both its ADP-ribosyl cyclase and the base exchange activities were reduced by 10,000- and 18,000-fold, respectively. Substituting Glu(179) with either Asn, Leu, Asp, or Gln produced similar inactive enzymes, and so was the conversion of Trp(77) to Gly. However, both E179G and the double mutant E179G/W77G retained NAD-binding ability as shown by photoaffinity labeling with [(32)P]8-azido-NAD. These results indicate that both Glu(179) and Trp(77) are crucial for catalysis and

  19. Adenylyl cyclase subtype 1 is essential for late-phase long term potentiation and spatial propagation of synaptic responses in the anterior cingulate cortex of adult mice.

    PubMed

    Chen, Tao; O'Den, Gerile; Song, Qian; Koga, Kohei; Zhang, Ming-Ming; Zhuo, Min

    2014-10-10

    Long-term potentiation (LTP) is a key cellular mechanism for pathological pain in the central nervous system. LTP contains at least two different phases: early-phase LTP (E-LTP) and late-phase LTP (L-LTP). Among several major cortical areas, the anterior cingulate cortex (ACC) is a critical brain region for pain perception and its related emotional changes. Periphery tissue or nerve injuries cause LTP of excitatory synaptic transmission in the ACC. Our previous studies have demonstrated that genetic deletion of calcium-stimulated adenylyl cyclase 1 (AC1) or pharmacological application of a selective AC1 inhibitor NB001 blocked E-LTP in the ACC. However, the effect of AC1 on L-LTP, which requires new protein synthesis and is important for the process of chronic pain, has not been investigated. Here we tested the effects of NB001 on the ACC L-LTP and found that bath application of NB001 (0.1 μM) totally blocked the induction of L-LTP and recruitment of cortical circuitry without affecting basal excitatory transmission. In contrast, gabapentin, a widely used analgesic drug for neuropathic pain, did not block the induction of L-LTP and circuitry recruitment even at a high concentration (100 μM). Gabapentin non-selectively decreased basal synaptic transmission. Our results provide strong evidence that the selective AC1 inhibitor NB001 can be used to inhibit pain-related cortical L-LTP without affecting basal synaptic transmission. It also provides basic mechanisms for possible side effects of gabapentin in the central nervous system and its ineffectiveness in some patients with neuropathic pain.

  20. Asymmetrically acting lycopene beta-cyclases (CrtLm) from non-photosynthetic bacteria.

    PubMed

    Tao, L; Picataggio, S; Rouvière, P E; Cheng, Q

    2004-03-01

    Carotenoids have important functions in photosynthesis, nutrition, and protection against oxidative damage. Some natural carotenoids are asymmetrical molecules that are difficult to produce chemically. Biological production of carotenoids using specific enzymes is a potential alternative to extraction from natural sources. Here we report the isolation of lycopene beta-cyclases that selectively cyclize only one end of lycopene or neurosporene. The crtLm genes encoding the asymmetrically acting lycopene beta-cyclases were isolated from non-photosynthetic bacteria that produced monocyclic carotenoids. Co-expression of these crtLm genes with the crtEIB genes from Pantoea stewartii (responsible for lycopene synthesis) resulted in the production of monocyclic gamma-carotene in Escherichia coli. The asymmetric cyclization activity of CrtLm could be inhibited by the lycopene beta-cyclase inhibitor 2-(4-chlorophenylthio)-triethylamine (CPTA). Phylogenetic analysis suggested that bacterial CrtL-type lycopene beta-cyclases might represent an evolutionary link between the common bacterial CrtY-type of lycopene beta-cyclases and plant lycopene beta- and epsilon-cyclases. These lycopene beta-cyclases may be used for efficient production of high-value asymmetrically cyclized carotenoids.

  1. Guanylyl cyclase structure, function and regulation.

    PubMed

    Potter, Lincoln R

    2011-12-01

    Nitric oxide, bicarbonate, natriuretic peptides (ANP, BNP and CNP), guanylins, uroguanylins and guanylyl cyclase activating proteins (GCAPs) activate a family of enzymes variously called guanyl, guanylyl or guanylate cyclases that catalyze the conversion of guanosine triphosphate to cyclic guanosine monophosphate (cGMP) and pyrophosphate. Intracellular cyclic GMP is a second messenger that modulates: platelet aggregation, neurotransmission, sexual arousal, gut peristalsis, blood pressure, long bone growth, intestinal fluid secretion, lipolysis, phototransduction, cardiac hypertrophy and oocyte maturation. This review briefly discusses the discovery of cGMP and guanylyl cyclases, then nitric oxide, nitric oxide synthase and soluble guanylyl cyclase are described in slightly greater detail. Finally, the structure, function, and regulation of the individual mammalian single membrane-spanning guanylyl cyclases GC-A, GC-B, GC-C, GC-D, GC-E, GC-F and GC-G are described in greatest detail as determined by biochemical, cell biological and gene-deletion studies.

  2. Dephosphorylation of sperm guanylate cyclase during sea urchin fertilization

    SciTech Connect

    Ward, G.E.

    1985-01-01

    When intact Arbacia punctulata spermatozoa are exposed to solubilized egg jelly, the electrophoretic mobility of an abundant sperm flagellar membrane protein changes from an apparent molecular mass of 160 kDa to 150 kDa. A. punctulata spermatozoa can be labeled in vivo with /sup 32/P-labeled cells it was demonstrated that the mobility shift of the 160-kDa protein is due to dephosphorylation. The peptide resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-Gly-Gly-Gly-Arg-Leu-NH/sub 2/) is the component of egg jelly which is responsible for inducing the dephosphorylation. The 160/150-kdal sperm membrane protein has been purified to homogeneity by affinity chromatography on concanavalin A-agarose, and identified as sperm guanylate cyclase. The enzymatic activity of the guanylate cyclase is tightly coupled to its phosphorylation state. Resact has been shown to act as a potent chemoattractant for A. punctulata spermatozoa. The chemotactic response is concentration-dependent, is abolished by pretreatment of the spermatozoa with resact, and shows an absolute requirement for external calcium. This work represents the first demonstration of animal sperm chemotaxis in response to a precisely-defined molecule of egg origin. The results established a new, biologically meaningful function for resact, and may implicate sperm guanylate cyclase and cGMP in flagellar function and the chemotactic response.

  3. Discovery and Characterization of a New Family of Diterpene Cyclases in Bacteria and Fungi.

    PubMed

    Yang, Yan-Long; Zhang, Shasha; Ma, Ke; Xu, Yuxing; Tao, Qiaoqiao; Chen, Yihua; Chen, Juan; Guo, Shunxing; Ren, Jinwei; Wang, Wenzhao; Tao, Yong; Yin, Wen-Bing; Liu, Hongwei

    2017-04-18

    Diterpene cyclases from bacteria and basidiomycete fungi are seldom studied. Here, we presented the identification and verification of EriG, a member of the UbiA superfamily, as the enzyme responsible for the cyclization of the cyathane skeleton in the mushroom Hericium erinaceum. Genome mining using the EriG protein sequence as a probe led to the discovery of a new family of ubiquitous UbiA-related diterpene cyclases in bacteria and fungi. We successfully characterized seven new diterpene cyclases from bacteria or basidiomycete fungi with the help of an engineered Escherichia coli strain and determined the structures of their corresponding products. A new diterpene with an unusual skeleton was generated during this process. The discovery of this new family of diterpene cyclases provides new insight into the UbiA superfamily.

  4. [Biosynthesis of cyclic GMP in plant cells - new insight into guanylate cyclases].

    PubMed

    Świeżawska, Brygida; Marciniak, Katarzyna; Szmidt-Jaworska, Adriana

    2015-01-01

    Cyclic 3',5'-guanosine monophosphate (cGMP) is involved in many physiological processes in plants. Concentration of this second messenger in plant cell is determined by guanylyl cyclases (GCs) responsible for cGMP synthesis and phosphodiesterases (PDEs) involved in cGMP inactivation. First discovered plant GCs were localized in cytosol, but few years ago a new family of plasma membrane proteins with guanylyl cyclase activity was identified in Arabidopsis thaliana. These proteins belong to the family of a leucine-rich repeat receptor-like kinases (LRR-RLK) with extracellular leucine-rich repeat domain, a transmembrane-spanning domain, and an intracellular kinase domain. A novel class of guanylyl cyclases contain the GC catalytic center encapsulated within the intracellular kinase domain. These molecules are different to animal GCs in that the GC catalytic center is nested within the kinase domain. In presented paper we summarized the most recent data concerning plant guanylyl cyclases.

  5. Vasodilator responses to dopamine in rat perfused mesentery are age-dependent.

    PubMed Central

    Wanstall, J. C.; O'Donnell, S. R.

    1989-01-01

    1. Dose-dependent vasodilator responses to dopamine, isoprenaline, noradrenaline, 3-isobutyl-1-methylxanthine (IBMX) and sodium nitroprusside were obtained in isolated perfused mesentery preparations, taken from reserpine-treated rats of different ages. The preparations were pretreated with phenoxybenzamine (1 microM) and perfused with physiological salt solution containing cocaine (10 microM), additional KCl (20 mM) and vasopressin (0.1 microM). 2. Vasodilator responses to dopamine were abolished by the dopamine1 (DA1)-selective antagonist SCH 23390 (10 nM) and those to isoprenaline by propranolol (1 microM), but the vasodilator responses to noradrenaline were abolished only when SCH 23390 and propranolol were used together. This indicated that dopamine was acting via DA1-receptors, isoprenaline via beta-adrenoceptors and that noradrenaline could act via DA1-receptors and beta-adrenoceptors in this preparation. 3. Responses to all the vasodilator drugs decreased in magnitude between the ages of 1 and 2 months. Responses to dopamine declined further in 4 month-old rats and were negligible at 6 or 22-24 months of age. Responses to isoprenaline were well maintained up to 6 months of age, but were negligible at 22-24 months. 4. It is concluded that, in the rat mesenteric vasculature, there is a non-specific decline in responses to vasodilator drugs during development (1 to 2 months). Subsequently there is a specific decline in DA1-receptor-mediated and beta-adrenoceptor-mediated responses; the former are lost at an earlier age than the latter. This different time course suggests that age influences receptor numbers, or their coupling to adenylate cyclase, rather than a post-receptor event in the adenylate cyclase/cyclic AMP pathway. PMID:2804550

  6. A Novel Mechanism for Adenylyl Cyclase Inhibition from the Crystal Structure of its Complex with Catechol Estrogen

    SciTech Connect

    Steegborn,C.; Litvin, T.; Hess, K.; Capper, A.; Taussig, R.; Buck, J.; Levin, L.; Wu, H.

    2005-01-01

    Catechol estrogens are steroid metabolites that elicit physiological responses through binding to a variety of cellular targets. We show here that catechol estrogens directly inhibit soluble adenylyl cyclases and the abundant trans-membrane adenylyl cyclases. Catechol estrogen inhibition is non-competitive with respect to the substrate ATP, and we solved the crystal structure of a catechol estrogen bound to a soluble adenylyl cyclase from Spirulina platensis in complex with a substrate analog. The catechol estrogen is bound to a newly identified, conserved hydrophobic patch near the active center but distinct from the ATP-binding cleft. Inhibitor binding leads to a chelating interaction between the catechol estrogen hydroxyl groups and the catalytic magnesium ion, distorting the active site and trapping the enzyme substrate complex in a non-productive conformation. This novel inhibition mechanism likely applies to other adenylyl cyclase inhibitors, and the identified ligand-binding site has important implications for the development of specific adenylyl cyclase inhibitors.

  7. Diversity of Structure and Function of Response Regulator Output Domains

    PubMed Central

    Galperin, Michael Y.

    2011-01-01

    Summary Response regulators (RRs) within two-component signal transduction systems control a variety of cellular processes. Most RRs contain DNA-binding output domains and serve as transcriptional regulators. Other RR types contain RNA-binding, ligand-binding, protein-binding or transporter output domains and exert regulation at the transcriptional, post-transcriptional or post-translational levels. In a significant fraction of RRs, output domains are enzymes that themselves participate in signal transduction: methylesterases, adenylate or diguanylate cyclases, c-di-GMP-specific phosphodiesterases, histidine kinases, serine/threonine protein kinases and protein phosphatases. In addition, there remain output domains whose functions are still unknown. Patterns of the distribution of various RR families are generally conserved within key microbial lineages and can be used to trace adaptations of various species to their unique ecological niches. PMID:20226724

  8. Diversity of structure and function of response regulator output domains.

    PubMed

    Galperin, Michael Y

    2010-04-01

    Response regulators (RRs) within two-component signal transduction systems control a variety of cellular processes. Most RRs contain DNA-binding output domains and serve as transcriptional regulators. Other RR types contain RNA-binding, ligand-binding, protein-binding or transporter output domains and exert regulation at the transcriptional, post-transcriptional or post-translational levels. In a significant fraction of RRs, output domains are enzymes that themselves participate in signal transduction: methylesterases, adenylate or diguanylate cyclases, c-di-GMP-specific phosphodiesterases, histidine kinases, serine/threonine protein kinases and protein phosphatases. In addition, there remain output domains whose functions are still unknown. Patterns of the distribution of various RR families are generally conserved within key microbial lineages and can be used to trace adaptations of various species to their unique ecological niches.

  9. Mechanistic investigations on six bacterial terpene cyclases

    PubMed Central

    Rabe, Patrick; Schmitz, Thomas

    2016-01-01

    Summary The products obtained by incubation of farnesyl diphosphate (FPP) with six purified bacterial terpene cyclases were characterised by one- and two-dimensional NMR spectroscopic methods, allowing for a full structure elucidation. The absolute configurations of four terpenes were determined based on their optical rotary powers. Incubation experiments with 13C-labelled isotopomers of FPP in buffers containing water or deuterium oxide allowed for detailed insights into the cyclisation mechanisms of the bacterial terpene cyclases. PMID:27829890

  10. Linkage between Fitness of Yeast Cells and Adenylate Kinase Catalysis

    PubMed Central

    Tükenmez, Hasan; Magnussen, Helge Magnus; Kovermann, Michael; Byström, Anders; Wolf-Watz, Magnus

    2016-01-01

    Enzymes have evolved with highly specific values of their catalytic parameters kcat and KM. This poses fundamental biological questions about the selection pressures responsible for evolutionary tuning of these parameters. Here we are address these questions for the enzyme adenylate kinase (Adk) in eukaryotic yeast cells. A plasmid shuffling system was developed to allow quantification of relative fitness (calculated from growth rates) of yeast in response to perturbations of Adk activity introduced through mutations. Biophysical characterization verified that all variants studied were properly folded and that the mutations did not cause any substantial differences to thermal stability. We found that cytosolic Adk is essential for yeast viability in our strain background and that viability could not be restored with a catalytically dead, although properly folded Adk variant. There exist a massive overcapacity of Adk catalytic activity and only 12% of the wild type kcat is required for optimal growth at the stress condition 20°C. In summary, the approach developed here has provided new insights into the evolutionary tuning of kcat for Adk in a eukaryotic organism. The developed methodology may also become useful for uncovering new aspects of active site dynamics and also in enzyme design since a large library of enzyme variants can be screened rapidly by identifying viable colonies. PMID:27642758

  11. Cellular Responses to Beta Blocker Exposures in Marine Bivalves

    EPA Science Inventory

    β blockers are prescription drugs used for medical treatment of hypertension and arrhythmias. They prevent activation of adenylate cyclase and increases in blood pressure by limiting cAMP production and protein kinase A activation. After being taken therapeutically, β b...

  12. Coupled ATPase-adenylate kinase activity in ABC transporters

    PubMed Central

    Kaur, Hundeep; Lakatos-Karoly, Andrea; Vogel, Ramona; Nöll, Anne; Tampé, Robert; Glaubitz, Clemens

    2016-01-01

    ATP-binding cassette (ABC) transporters, a superfamily of integral membrane proteins, catalyse the translocation of substrates across the cellular membrane by ATP hydrolysis. Here we demonstrate by nucleotide turnover and binding studies based on 31P solid-state NMR spectroscopy that the ABC exporter and lipid A flippase MsbA can couple ATP hydrolysis to an adenylate kinase activity, where ADP is converted into AMP and ATP. Single-point mutations reveal that both ATPase and adenylate kinase mechanisms are associated with the same conserved motifs of the nucleotide-binding domain. Based on these results, we propose a model for the coupled ATPase-adenylate kinase mechanism, involving the canonical and an additional nucleotide-binding site. We extend these findings to other prokaryotic ABC exporters, namely LmrA and TmrAB, suggesting that the coupled activities are a general feature of ABC exporters. PMID:28004795

  13. Genetic and biochemical analysis of the adenylyl cyclase-associated protein, cap, in Schizosaccharomyces pombe.

    PubMed Central

    Kawamukai, M; Gerst, J; Field, J; Riggs, M; Rodgers, L; Wigler, M; Young, D

    1992-01-01

    We have identified, cloned, and studied a gene, cap, encoding a protein that is associated with adenylyl cyclase in the fission yeast Schizosaccharomyces pombe. This protein shares significant sequence homology with the adenylyl cyclase-associated CAP protein in the yeast Saccharomyces cerevisiae. CAP is a bifunctional protein; the N-terminal domain appears to be involved in cellular responsiveness to RAS, whereas loss of the C-terminal portion is associated with morphological and nutritional defects. S. pombe cap can suppress phenotypes associated with deletion of the C-terminal CAP domain in S. cerevisiae but does not suppress phenotypes associated with deletion of the N-terminal domain. Analysis of cap disruptants also mapped the function of cap to two domains. The functional loss of the C-terminal region of S. pombe cap results in abnormal cellular morphology, slow growth, and failure to grow at 37 degrees C. Increases in mating and sporulation were observed when the entire gene was disrupted. Overproduction of both cap and adenylyl cyclase results in highly elongated large cells that are sterile and have measurably higher levels of adenylyl cyclase activity. Our results indicate that cap is required for the proper function of S. pombe adenylyl cyclase but that the C-terminal domain of cap has other functions that are shared with the C-terminal domain of S. cerevisiae CAP. Images PMID:1550959

  14. 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.

  15. Age-related decline of autocrine pituitary adenylate cyclase-activating polypeptide impairs angiogenic capacity of rat cerebromicrovascular endothelial cells.

    PubMed

    Banki, Eszter; Sosnowska, Danuta; Tucsek, Zsuzsanna; Gautam, Tripti; Toth, Peter; Tarantini, Stefano; Tamas, Andrea; Helyes, Zsuzsanna; Reglodi, Dora; Sonntag, William E; Csiszar, Anna; Ungvari, Zoltan

    2015-06-01

    Aging impairs angiogenic capacity of cerebromicrovascular endothelial cells (CMVECs) promoting microvascular rarefaction, but the underlying mechanisms remain elusive. PACAP is an evolutionarily conserved neuropeptide secreted by endothelial cells and neurons, which confers important antiaging effects. To test the hypothesis that age-related changes in autocrine PACAP signaling contributes to dysregulation of endothelial angiogenic capacity, primary CMVECs were isolated from 3-month-old (young) and 24-month-old (aged) Fischer 344 x Brown Norway rats. In aged CMVECs, expression of PACAP was decreased, which was associated with impaired capacity to form capillary-like structures, impaired adhesiveness to collagen (assessed using electric cell-substrate impedance sensing [ECIS] technology), and increased apoptosis (caspase3 activity) when compared with young cells. Overexpression of PACAP in aged CMVECs resulted in increased formation of capillary-like structures, whereas it did not affect cell adhesion. Treatment with recombinant PACAP also significantly increased endothelial tube formation and inhibited apoptosis in aged CMVECs. In young CMVECs shRNA knockdown of autocrine PACAP expression significantly impaired tube formation capacity, mimicking the aging phenotype. Cellular and mitochondrial reactive oxygen species production (dihydroethidium and MitoSox fluorescence, respectively) were increased in aged CMVECs and were unaffected by PACAP. Collectively, PACAP exerts proangiogenic effects and age-related dysregulation of autocrine PACAP signaling may contribute to impaired angiogenic capacity of CMVECs in aging.

  16. Age-Related Decline of Autocrine Pituitary Adenylate Cyclase-Activating Polypeptide Impairs Angiogenic Capacity of Rat Cerebromicrovascular Endothelial Cells

    PubMed Central

    Banki, Eszter; Sosnowska, Danuta; Tucsek, Zsuzsanna; Gautam, Tripti; Toth, Peter; Tarantini, Stefano; Tamas, Andrea; Helyes, Zsuzsanna; Reglodi, Dora; Sonntag, William E.; Csiszar, Anna

    2015-01-01

    Aging impairs angiogenic capacity of cerebromicrovascular endothelial cells (CMVECs) promoting microvascular rarefaction, but the underlying mechanisms remain elusive. PACAP is an evolutionarily conserved neuropeptide secreted by endothelial cells and neurons, which confers important antiaging effects. To test the hypothesis that age-related changes in autocrine PACAP signaling contributes to dysregulation of endothelial angiogenic capacity, primary CMVECs were isolated from 3-month-old (young) and 24-month-old (aged) Fischer 344 x Brown Norway rats. In aged CMVECs, expression of PACAP was decreased, which was associated with impaired capacity to form capillary-like structures, impaired adhesiveness to collagen (assessed using electric cell-substrate impedance sensing [ECIS] technology), and increased apoptosis (caspase3 activity) when compared with young cells. Overexpression of PACAP in aged CMVECs resulted in increased formation of capillary-like structures, whereas it did not affect cell adhesion. Treatment with recombinant PACAP also significantly increased endothelial tube formation and inhibited apoptosis in aged CMVECs. In young CMVECs shRNA knockdown of autocrine PACAP expression significantly impaired tube formation capacity, mimicking the aging phenotype. Cellular and mitochondrial reactive oxygen species production (dihydroethidium and MitoSox fluorescence, respectively) were increased in aged CMVECs and were unaffected by PACAP. Collectively, PACAP exerts proangiogenic effects and age-related dysregulation of autocrine PACAP signaling may contribute to impaired angiogenic capacity of CMVECs in aging. PMID:25136000

  17. Properties of adenyl cyclase and cyclic adenosine 3',5'-monophosphate receptor protein-deficient mutants of Escherichia coli.

    PubMed Central

    Kumar, S

    1976-01-01

    Several spontaneous cya and crp mutants of Escherichia coli have been selected as clones simultaneously resistant to phage lambda and nalidixic acid and characterized. Both cya and crp mutants have been found to grow as cocci with increased doubling times. They have increased resistance to some mutagens (methylmethanesulfonate, ultraviolet light, gamma rays), antibiotics (nalidixic acid, ampicillin), phages (lambda, T6), sublethal heat and hypotonic shock, and decreased resistance to neutral detergents (sodium dodecyl sulfate, sodium deoxycholate), a protein synthesis inhibitor (streptomycin), and a respiratory inhibitor (sodium azide). The nature of changes in cell parameters indicate fundamental alterations in the envelope structure of the cya and crp mutant cells. The new cya and crp mutants have been found to be multiply carbohydrate negative and nonmotile in conformity with similar previously isolated mutants. Studies of revertants and phi80 cya+ and phi80 cya transductants indicated that the pleiotropic phenotype is related to a single mutational event at the cya or the crp locus in the mutants. Images PMID:173710

  18. Adenylation by testis-specific cytoplasmic poly(A) polymerase, PAPOLB/TPAP, is essential for spermatogenesis

    PubMed Central

    KASHIWABARA, Shin-ichi; TSURUTA, Satsuki; OKADA, Keitaro; YAMAOKA, Yutaro; BABA, Tadashi

    2016-01-01

    The testis-specific cytoplasmic poly(A) polymerase PAPOLB/TPAP is essential for spermatogenesis. Although this enzyme is responsible for poly(A) tail extension of a subset of mRNAs in round spermatids, the stability and translational efficiency of these mRNAs are unaffected by the absence of PAPOLB. To clarify the functional importance of this enzyme’s adenylation activity, we produced PAPOLB-null mice expressing a polyadenylation-defective PAPOLB mutant (PAPOLBD114A), in which the catalytic Asp at residue 114 was mutated to Ala. Introducing PAPOLBD114A failed to rescue PAPOLB-null phenotypes, such as reduced expression of haploid-specific mRNAs, spermiogenesis arrest, and male infertility. These results suggest that PAPOLB regulates spermatogenesis through its adenylation activity. PMID:27647534

  19. Different effect of prostaglandin E2 on B-cell activation by two distinct B-cell differentiation factors, B151-TRF1/IL-5 and B151-TRF2: selective inhibition of B151-TRF2-induced antibody response through increases in intracellular cyclic AMP levels

    PubMed Central

    Ishihara, K.; Ono, S.; Takahama, Y.; Hirayama, F.; Hirano, H.; Itoh, K.; Dobashi, K.; Murakami, S.; Katoh, Y.; Yamaguchi, M.; Hamaoka, T.

    1989-01-01

    Effects of prostaglandin E2 (PGE2) on murine B-cell activation induced by two distinct B-cell differentiation factors, B151-TRF1/IL-5 and B151-TRF2, were examined. A final differentiation of unprimed B cells into IgM-producing cells induced by B151-TRF2 was markedly inhibited by PGE2 at physiological concentrations (around 10-8 M), whereas B151-TRF1/IL-5-induced antibody responses of unprimed as well as activated B cells were not affected by PGE2, even at 10-6 M. B-cell responses induced by B151-TRF2-like factors from autoimmune-prone MRL/1pr mice were also inhibited by PGE2. Biphasic increases in intracellular cyclic AMP (cAMP) levels were induced by culturing B cells with 10-6 or 10-8 M PGE2: rapid increases within 8 min and delayed increases around 16 hr. The direct addition of dibutyryl cAMP to cultures of B cells resulted in marked inhibition of antibody responses when stimulated with B151-TRF2 but not with B151-TRF1/IL-5. The B151-TRF2-induced antibody responses were also inhibited by cAMP-elevating reagents such as forskolin, cholera toxin and theophyline. Furthermore, 2′, 5′-dideoxyadenosine, which is an inhibitor of adenylate cyclase, prevented the PGE2-mediated cAMP accumulation in unprimed B cells as well as the PGE2-mediated inhibition of B151-TRF2-induced B-cell responses when added at the initiation of culture. These results suggest that PGE2 inhibits B151-TRF2-induced antibody responses through the activation of adenylate cyclase and subsequent accumulation of intracellular cAMP, whereas B151-TRF1/IL-5-responsive B cells are resistant to the inhibitory effect of PGE2 and cAMP. PMID:2553585

  20. [Potentiation of nitric oxide-dependent activation of soluble guanylate cyclase by levomycetin, tetracycline, and oxolin].

    PubMed

    Shchegolev, A Iu; Sidorova, T A; Severina, I S

    2009-01-01

    The influence of antibiotics laevomycetin and tetracycline and the antivirus agent oxolin on the activity of human platelet soluble guanylate cyclase, the stimulation of the enzyme by NO-donors (sodium nitroprusside (SNP) and spermine nanoate (spermine NONO)) and the combination of spermine NONO and YC-1 was investigated. All preparations used in the concentration range 0,1-10 mM had no effect on the basal activity of guanylate cyclase but potentiated the SNP-induced activation of this enzyme. All preparations used synergistically increased (similar to YC-1) spermine NONO-induced activation of soluble guanylate cyclase. At the same time these compounds did not produce the leftward shift of spermine NONO concentration response curve characteristic for YC-1. Moreover, all compounds used did not influence the leftward shift of spermine NONO concentration response curve obtained in the presence of YC-1. This demonstrated that there was no competition between YC-1 and the drugs for interaction with the enzyme. The revealed regulatory phenomen of laevomycetin, tetracycline and oxolin to increase synergistically NO-dependent activation of soluble guanylate cyclase may cause additional pharmacological effects during prolong treatment by these drugs. This fact is necessary taking into account.

  1. Membrane guanylyl cyclase receptors: an update

    PubMed Central

    Garbers, David L.; Chrisman, Ted D.; Wiegn, Phi; Katafuchi, Takeshi; Albanesi, Joseph P.; Bielinski, Vincent; Barylko, Barbara; Redfield, Margaret M.; Burnett, John C.

    2007-01-01

    Recent studies have demonstrated key roles for several membrane guanylyl cyclase receptors in the regulation of cell hyperplasia, hypertrophy, migration and extracellular matrix production, all of which having an impact on clinically relevant diseases, including tissue remodeling after injury. Additionally, cell differentiation, and even tumor progression, can be profoundly influenced by one or more of these receptors. Some of these receptors also mediate important communication between the heart and intestine, and the kidney to regulate blood volume and Na+ balance. PMID:16815030

  2. The energy landscape of adenylate kinase during catalysis

    PubMed Central

    Kerns, S. Jordan; Agafonov, Roman V.; Cho, Young-Jin; Pontiggia, Francesco; Otten, Renee; Pachov, Dimitar V.; Kutter, Steffen; Phung, Lien A.; Murphy, Padraig N.; Thai, Vu; Alber, Tom; Hagan, Michael F.; Kern, Dorothee

    2014-01-01

    Kinases perform phosphoryl-transfer reactions in milliseconds; without enzymes, these reactions would take about 8000 years under physiological conditions. Despite extensive studies, a comprehensive understanding of kinase energy landscapes, including both chemical and conformational steps, is lacking. Here we scrutinize the microscopic steps in the catalytic cycle of adenylate kinase, through a combination of NMR measurements during catalysis, pre-steady-state kinetics, MD simulations, and crystallography of active complexes. We find that the Mg2+ cofactor activates two distinct molecular events, phosphoryl transfer (>105-fold) and lid-opening (103-fold). In contrast, mutation of an essential active-site arginine decelerates phosphoryl transfer 103-fold without substantially affecting lid-opening. Our results highlight the importance of the entire energy landscape in catalysis and suggest that adenylate kinases have evolved to activate key processes simultaneously by precise placement of a single, charged and very abundant cofactor in a pre-organized active site. PMID:25580578

  3. Aminoacyl transfer from an adenylate anhydride to polyribonucleotides

    NASA Technical Reports Server (NTRS)

    Weber, A. L.; Lacey, J. C., Jr.

    1975-01-01

    Imidazole catalysis of phenylalanyl transfer from phenylalanine adenylate to hydroxyl groups of homopolyribonucleotides is studied as a possible chemical model of biochemical aminoacylation of transfer RNA (tRNA). The effect of pH on imidazole-catalyzed transfer of phenylalanyl residues to poly(U) and poly(A) double helix strands, the number of peptide linkages and their lability to base and neutral hydroxylamine, and the nature of adenylate condensation products are investigated. The chemical model entertained exhibits a constraint by not acylating the hydroxyl groups of polyribonucleotides in a double helix. The constraint is consistent with selective biochemical aminoacylation at the tRNA terminus. Interest in imidazole as a model of histidine residue in protoenzymes participating in prebiotic aminoacyl transfer to polyribonucleotides, and in rendering the tRNA a more efficient adaptor, is indicated.

  4. Functional and Phylogenetic Divergence of Fungal Adenylate-Forming Reductases

    PubMed Central

    Kalb, Daniel; Lackner, Gerald

    2014-01-01

    A key step in fungal l-lysine biosynthesis is catalyzed by adenylate-forming l-α-aminoadipic acid reductases, organized in domains for adenylation, thiolation, and the reduction step. However, the genomes of numerous ascomycetes and basidiomycetes contain an unexpectedly large number of additional genes encoding similar but functionally distinct enzymes. Here, we describe the functional in vitro characterization of four reductases which were heterologously produced in Escherichia coli. The Ceriporiopsis subvermispora serine reductase Nps1 features a terminal ferredoxin-NADP+ reductase (FNR) domain and thus belongs to a hitherto undescribed class of fungal multidomain enzymes. The second major class is characterized by the canonical terminal short-chain dehydrogenase/reductase domain and represented by Ceriporiopsis subvermispora Nps3 as the first biochemically characterized l-α-aminoadipic acid reductase of basidiomycete origin. Aspergillus flavus l-tyrosine reductases LnaA and LnbA are members of a distinct phylogenetic clade. Phylogenetic analysis supports the view that fungal adenylate-forming reductases are more diverse than previously recognized and belong to four distinct classes. PMID:25085485

  5. Glucose and GLP-1 Stimulate cAMP Production via Distinct Adenylyl Cyclases in INS-1E Insulinoma Cells

    PubMed Central

    Ramos, Lavoisier S.; Zippin, Jonathan Hale; Kamenetsky, Margarita; Buck, Jochen; Levin, Lonny R.

    2008-01-01

    In β cells, both glucose and hormones, such as GLP-1, stimulate production of the second messenger cAMP, but glucose and GLP-1 elicit distinct cellular responses. We now show in INS-1E insulinoma cells that glucose and GLP-1 produce cAMP with distinct kinetics via different adenylyl cyclases. GLP-1 induces a rapid cAMP signal mediated by G protein–responsive transmembrane adenylyl cyclases (tmAC). In contrast, glucose elicits a delayed cAMP rise mediated by bicarbonate, calcium, and ATP-sensitive soluble adenylyl cyclase (sAC). This glucose-induced, sAC-dependent cAMP rise is dependent upon calcium influx and is responsible for the glucose-induced activation of the mitogen-activated protein kinase (ERK1/2) pathway. These results demonstrate that sAC-generated and tmAC-generated cAMP define distinct signaling cascades. PMID:18695009

  6. Differential expression of functional guanylyl cyclases in melanocytes: absence of nitric-oxide-sensitive isoform in metastatic cells.

    PubMed

    Ivanova, K; Das, P K; van den Wijngaard, R M; Lenz, W; Klockenbring, T; Malcharzyk, V; Drummer, C; Gerzer, R

    2001-03-01

    Nitric oxide (NO) is a reactive endogenous molecule with multiple functions and its cellular signaling activity is mainly mediated by activation of the soluble isoform of guanylyl cyclase, a heterodimeric (alpha/beta) hemeprotein. The expression of the NO-sensitive soluble isoform of guanylyl cyclase was studied in various cultured melanocytic cells by measuring the accumulation of guanosine 3',5'-cyclic monophosphate in the presence and absence of NO donors. Here we report that 3-morpholino-sydnonimine, a donor of NO redox species, and (Z)-1-[2- (2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate, a direct NO donor, induced a 20-fold increase in intracellular guanosine 3',5'-cyclic monophosphate in nonmetastatic melanoma cells and normal melanocytes in culture that could be related to cellular melanin content in a concentration-dependent manner. The increased intracellular guanosine 3',5'-cyclic monophosphate was due to stimulation of the activity of soluble guanylyl cyclase as such increase was completely abolished by using a specific inhibitor of soluble guanylyl cyclase. The involvement of functional soluble guanylyl cyclase was further confirmed by the presence of alpha1 and beta1 subunits in these cells at both mRNA and protein levels. In contrast, none of the NO donors induced guanosine 3',5'-cyclic monophosphate production in metastatic melanoma cells, which could be attributed to the absence of the beta1 subunit that is essential for catalytic activity of the soluble isoform of guanylyl cyclase. Metastatic melanoma cells produced higher levels of intracellular guanosine 3',5'-cyclic monophosphate in response to natriuretic peptides than other cell types, however, due to upregulation of membrane-bound guanylyl cyclase activities, but they are less pigmented or unpigmented. The present finding suggests that NO signaling in association with melanogenesis is dependent on the soluble isoform of guanylyl cyclase, whereas absence of soluble guanylyl

  7. [Potentiation of NO-dependent activation of soluble guanylyl cyclase by 5-nitroisatin and antiviral preparatation arbidol].

    PubMed

    Severina, I S; Shchegolev, A Iu; Medvedev, A E

    2013-01-01

    Isatin (indole-dione) is an endogenous indole that exibits a wide range of biological and physiological activity. The influence of isatin derivatives 5-nitroisatin and arbidol (an antiviral preparatation) on spermine NONO-induced activation of human platelet soluble guanylyl cyclase was investigated. 5-nitroistnin and arbidol had no effect on basal activity, but synergistically increased in a concentration-dependent manner the spermine NONO-induced activation of this enzyme. 5-Nitroisatin and arbidol, like YC-1, sensitized guanylyl cyclase towards nitric oxide (NO) and produced a leftward shift of the spermine NONO concentration response curve. At the same time both compounds used did not influence the activation of guanylyl cyclase by YC-1 and did not change the synergistic increase of spermine NONO-induced activation of soluble guanylyl cyclase in the presence of YC-1. This suggests that 5-nitroisanin and arbidol did not compete with YC-1. Addition of isatin did not change the synergistic increase in the spermine NONO-induced guanylyl cyclase activation by 5-nitroisatin and arbidol and did not influence a leftward shift of spermine NONO concentration response curve produced by these compounds. These data suggest lack of competitive interaction between isatin and both its derivatives used.

  8. An aberrant adenylate kinase isoenzyme from the serum of patients with Duchenne muscular dystrophy.

    PubMed

    Hamada, M; Okuda, H; Oka, K; Watanabe, T; Ueda, K; Nojima, M; Kuby, S A; Manship, M; Tyler, F H; Ziter, F A

    1981-08-13

    The sera from patients with human Duchenne (X-linked) progressive muscular dystrophy contain elevated adenylate kinase (ATP: AMP phosphotransferase, EC 2.7.4.3) activities, in addition to their characteristically high creatine kinase (ATP; creatine N-phosphotransferase, EC 2.7.3.2) activities. By agarose gel electrophoresis of human Duchenne dystrophic serum, the presence of an apparently normal human serum adenylate kinase together with a variant species of adenylate kinase was detected. The latter enzyme species appeared, in its mobility, to be similar to that of the normal human liver-type adenylate kinase. The presence of this aberrant liver-type adenylate kinase could also be demonstrated by characteristic (for the liver type) inhibition patterns with P1,P5-di-(adenosine-5')pentaphosphate, 5,5'-dithiobis(2-nitrobenzoate) and phosphoenolpyruvate. On the other hand, by inhibition titrations with an anti-muscle-type adenylate kinase, hemolysates from the erythrocytes of several Duchenne and Becker's dystrophics were found to contain approx. 96% muscle-type adenylate kinase and their serum approx. 97% muscle-type adenylate kinase. These same patients contained approx. 89% M-M type creatine kinase in their serum (by inhibition against anti-human muscle-type creatine kinase) indicative of the presence also of M-B plus B-B type active isoenzymes. All of these data can best be explained by the presence of a variant or mutant adenylate kinase isoenzyme in the dystrophic serum. This isoenzyme appears to resemble the liver type in its inhibition patterns with P1,P5-di(adenosine-5')pentaphosphate, 5,5'-dithiobis(2-nitrobenzoate) and phosphoenolpyruvate, and in its heat stability (compare also the agarose gel electrophoresis pattern); but structurally, it is a muscle type, or derived from a muscle type, as shown immunologically by inhibition reactions with anti-muscle-type adenylate kinase. Whether this is a fetal-type isoenzyme of adenylate kinase will require further

  9. Isoform-targeted regulation of cardiac adenylyl cyclase.

    PubMed

    Ishikawa, Yoshihiro

    2003-01-01

    Numerous attempts have been made to develop strategies for regulating the intracellular cyclic AMP signal pharmacologically, with an intention to establish either new medical therapeutic methods or experimental tools. In the past decades, many pharmacological reagents have been identified that regulate this pathway at the level of the receptor. G protein, adenylyl cyclase, cyclic AMP, protein kinase A and phosphodiesterase. Since the cloning of adenylyl cyclase isoforms during the 1990s, investigators including ourselves have tried to find reagents that regulate the activity of this enzyme directly in an isoform-dependent manner. The ultimate goal of developing such reagents would be to regulate the cyclic AMP signal in an organ-dependent manner. Ourselves and other workers have reported that such reagents may vary from a simple cation to kinases. In a more recent study, using the results from crystallographic studies and computer-assisted drug design programs, we have identified subtype-selective regulators of adenylyl cyclase. Such regulators are mostly based upon forskolin, a diterpene compound obtained from Coleus forskolii, that acts directly on adenylyl cyclase to increase the intracellular levels of cyclic AMP. Similarly, novel reagents have been identified that inhibit a specific adenylyl cyclase isoform (e.g. type 5 adenylyl cyclase). Such reagents would potentially provide a new therapeutic strategy to treat hypertension, for example, as well as methods to selectively stimulate or inhibit this adenylyl cyclase isoform, which may be reminiscent of overexpression or knocking out of the cardiac adenylyl cyclase isoform by the use of a pharmacological method.

  10. Crystallization of cyclase-associated protein from Dictyostelium discoideum.

    PubMed

    Hofmann, Andreas; Hess, Sonja; Noegel, Angelika A; Schleicher, Michael; Wlodawer, Alexander

    2002-10-01

    Cyclase-associated protein (CAP) is a conserved two-domain protein that helps to activate the catalytic activity of adenylyl cyclase in the cyclase-bound state through interaction with Ras, which binds to the cyclase in a different region. With its other domain, CAP can bind monomeric actin and therefore also carries a cytoskeletal function. The protein is thus involved in Ras/cAMP-dependent signal transduction and most likely serves as an adapter protein translocating the adenylyl cyclase complex to the actin cytoskeleton. Crystals belonging to the orthorhombic space group C222, with unit-cell parameters a = 71.2, b = 75.1, c = 162.9 A, have been obtained from Dictyostelium discoideum CAP carrying a C-terminal His tag. A complete native data set extending to 2.2 A resolution was collected from a single crystal using an in-house X-ray system. The asymmetric unit contains one molecule of CAP.

  11. The cyclase-associated protein CAP as regulator of cell polarity and cAMP signaling in Dictyostelium.

    PubMed

    Noegel, Angelika A; Blau-Wasser, Rosemarie; Sultana, Hameeda; Müller, Rolf; Israel, Lars; Schleicher, Michael; Patel, Hitesh; Weijer, Cornelis J

    2004-02-01

    Cyclase-associated protein (CAP) is an evolutionarily conserved regulator of the G-actin/F-actin ratio and, in yeast, is involved in regulating the adenylyl cyclase activity. We show that cell polarization, F-actin organization, and phototaxis are altered in a Dictyostelium CAP knockout mutant. Furthermore, in complementation assays we determined the roles of the individual domains in signaling and regulation of the actin cytoskeleton. We studied in detail the adenylyl cyclase activity and found that the mutant cells have normal levels of the aggregation phase-specific adenylyl cyclase and that receptor-mediated activation is intact. However, cAMP relay that is responsible for the generation of propagating cAMP waves that control the chemotactic aggregation of starving Dictyostelium cells was altered, and the cAMP-induced cGMP production was significantly reduced. The data suggest an interaction of CAP with adenylyl cyclase in Dictyostelium and an influence on signaling pathways directly as well as through its function as a regulatory component of the cytoskeleton.

  12. Adenylate Energy Charge in Escherichia coli During Growth and Starvation

    PubMed Central

    Chapman, Astrid G.; Fall, Lana; Atkinson, Daniel E.

    1971-01-01

    The value of the adenylate energy charge, [(adenosine triphosphate) + ½ (adenosine diphosphate)]/[(adenosine triphosphate) + (adenosine diphosphate) + (adenosine monophosphate)], in Escherichia coli cells during growth is about 0.8. During the stationary phase after cessation of growth, or during starvation in carbon-limited cultures, the energy charge declines slowly to a value of about 0.5, and then falls more rapidly. During the slow decline in energy charge, all the cells are capable of forming colonies, but a rapid fall in viability coincides with the steep drop in energy charge. These results suggest that growth can occur only at energy charge values above about 0.8, that viability is maintained at values between 0.8 and 0.5, and that cells die at values below 0.5. Tabulation of adenylate concentrations previously reported for various organisms and tissues supports the prediction, based on enzyme kinetic observations in vitro, that the energy charge is stabilized near 0.85 in intact metabolizing cells of a wide variety of types. PMID:4333317

  13. A new knock-in mouse model of l-DOPA-responsive dystonia

    PubMed Central

    Rose, Samuel J.; Yu, Xin Y.; Heinzer, Ann K.; Harrast, Porter; Fan, Xueliang; Raike, Robert S.; Thompson, Valerie B.; Pare, Jean-Francois; Weinshenker, David; Smith, Yoland; Jinnah, Hyder A.

    2015-01-01

    Abnormal dopamine neurotransmission is associated with many different genetic and acquired dystonic disorders. For instance, mutations in genes critical for the synthesis of dopamine, including GCH1 and TH cause l-DOPA-responsive dystonia. Despite evidence that implicates abnormal dopamine neurotransmission in dystonia, the precise nature of the pre- and postsynaptic defects that result in dystonia are not known. To better understand these defects, we generated a knock-in mouse model of l-DOPA-responsive dystonia (DRD) mice that recapitulates the human p.381Q>K TH mutation (c.1141C>A). Mice homozygous for this mutation displayed the core features of the human disorder, including reduced TH activity, dystonia that worsened throughout the course of the active phase, and improvement in the dystonia in response to both l-DOPA and trihexyphenidyl. Although the gross anatomy of the nigrostriatal dopaminergic neurons was normal in DRD mice, the microstructure of striatal synapses was affected whereby the ratio of axo-spinous to axo-dendritic corticostriatal synaptic contacts was reduced. Microinjection of l-DOPA directly into the striatum ameliorated the dystonic movements but cerebellar microinjections of l-DOPA had no effect. Surprisingly, the striatal dopamine concentration was reduced to ∼1% of normal, a concentration more typically associated with akinesia, suggesting that (mal)adaptive postsynaptic responses may also play a role in the development of dystonia. Administration of D1- or D2-like dopamine receptor agonists to enhance dopamine signalling reduced the dystonic movements, whereas administration of D1- or D2-like dopamine receptor antagonists to further reduce dopamine signalling worsened the dystonia, suggesting that both receptors mediate the abnormal movements. Further, D1-dopamine receptors were supersensitive; adenylate cyclase activity, locomotor activity and stereotypy were exaggerated in DRD mice in response to the D1-dopamine receptor agonist SKF

  14. Identification of a CO2 responsive regulon in Bordetella.

    PubMed

    Hester, Sara E; Lui, Minghsun; Nicholson, Tracy; Nowacki, Daryl; Harvill, Eric T

    2012-01-01

    Sensing the environment allows pathogenic bacteria to coordinately regulate gene expression to maximize survival within or outside of a host. Here we show that Bordetella species regulate virulence factor expression in response to carbon dioxide levels that mimic in vivo conditions within the respiratory tract. We found strains of Bordetella bronchiseptica that did not produce adenylate cyclase toxin (ACT) when grown in liquid or solid media with ambient air aeration, but produced ACT and additional antigens when grown in air supplemented to 5% CO(2). Transcriptome analysis and quantitative real time-PCR analysis revealed that strain 761, as well as strain RB50, increased transcription of genes encoding ACT, filamentous hemagglutinin (FHA), pertactin, fimbriae and the type III secretion system in 5% CO(2) conditions, relative to ambient air. Furthermore, transcription of cyaA and fhaB in response to 5% CO(2) was increased even in the absence of BvgS. In vitro analysis also revealed increases in cytotoxicity and adherence when strains were grown in 5% CO(2). The human pathogens B. pertussis and B. parapertussis also increased transcription of several virulence factors when grown in 5% CO(2), indicating that this response is conserved among the classical bordetellae. Together, our data indicate that Bordetella species can sense and respond to physiologically relevant changes in CO(2) concentrations by regulating virulence factors important for colonization, persistence and evasion of the host immune response.

  15. Conformational transitions of Adenylate Kinase: switching by cracking

    PubMed Central

    Whitford, Paul C.; Miyashita, Osamu; Levy, Yaakov; Onuchic, José N.

    2007-01-01

    Conformational heterogeneity in proteins is known to often be the key to their function. We present a coarse grained model to explore the interplay between protein structure, folding and function which is applicable to allosteric or non-allosteric proteins. We employ the model to study the detailed mechanism of the reversible conformational transition of Adenylate Kinase (AKE) between the open to the closed conformation, a reaction that is crucial to the protein’s catalytic function. We directly observe high strain energy which appears to be correlated with localized unfolding during the functional transition. This work also demonstrates that competing native interactions from the open and closed form can account for the large conformational transitions in AKE. We further characterize the conformational transitions with a new measure ΦFunc, and demonstrate that local unfolding may be due, in part, to competing intra-protein interactions. PMID:17217965

  16. 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.

  17. 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

  18. Immunohistochemical Localization of Guanylate Cyclase within Neurons of Rat Brain

    NASA Astrophysics Data System (ADS)

    Ariano, Marjorie A.; Lewicki, John A.; Brandwein, Harvey J.; Murad, Ferid

    1982-02-01

    The immunohistochemical localization of guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] has been examined in rat neocortex, caudate-putamen, and cerebellum by using specific monoclonal antibodies. Immunofluorescence could be seen within somata and proximal dendrites of neurons in these regions. A nuclear immunofluorescence reaction to guanylate cyclase was characteristically absent. The staining pattern for guanylate cyclase was coincident with previously described localizations of cyclic GMP immunofluorescence within medium spiny neurons of the caudate-putamen and pyramidal cells of the neocortex. Cerebellar guanylate cyclase immunoreactivity was primarily confined to Purkinje cells and their primary dendrites, similar to the pattern reported for cyclic GMP-dependent protein kinase localization. Guanylate cyclase immunofluorescence was abolished when the monoclonal antibodies were exposed to purified enzyme prior to incubation of the tissue slices or when control antibody was substituted for the primary antibody. Immunohistochemical localization of cyclic AMP in these same tissues was readily distinguished from that of guanylate cyclase or cyclic GMP, showing uniform fluorescence throughout the cell bodies of neurons and glial elements.

  19. Central role of soluble adenylyl cyclase and cAMP in sperm physiology

    PubMed Central

    Buffone, Mariano G.; Wertheimer, Eva V.; Visconti, Pablo E.; Krapf, Dario

    2014-01-01

    Cyclic adenosine 3′,5′-monophosphate (cAMP), the first second messenger to be described, plays a central role in cell signaling in a wide variety of cell types. Over the last decades, a wide body of literature addressed the different roles of cAMP in cell physiology, mainly in response to neurotransmitters and hormones. cAMP is synthesized by a wide variety of adenylyl cylases that can generally be grouped in two types: transmembrane adenylyl cyclase and soluble adenylyl cyclases. In particular, several aspects of sperm physiology are regulated by cAMP produced by a single atypical adenylyl cyclase (Adcy10, aka sAC, SACY). The signature that identifies sAC among other ACs, is their direct stimulation by bicarbonate. The essential nature of cAMP in sperm function has been demonstrated using gain of function as well as loss of function approaches. This review unifies state of the art knowledge of the role of cAMP and those enzymes involved in cAMP signaling pathways required for the acquisition of fertilizing capacity of mammalian sperm. PMID:25066614

  20. Cyclase-associated proteins: CAPacity for linking signal transduction and actin polymerization.

    PubMed

    Hubberstey, Andrew V; Mottillo, Emilio P

    2002-04-01

    Many extracellular signals elicit changes in the actin cytoskeleton, which are mediated through an array of signaling proteins and pathways. One family of proteins that plays a role in regulating actin remodeling in response to cellular signals are the cyclase-associated proteins (CAPs). CAPs are highly conserved monomeric actin binding proteins present in a wide range of organisms including yeast, fly, plants, and mammals. The original CAP was isolated as a component of the Saccharomyces cerevisiae adenylyl cyclase complex that serves as an effector of Ras during nutritional signaling. CAPs are multifunctional molecules that contain domains involved in actin binding, adenylyl cyclase association in yeast, SH3 binding, and oligomerization. Genetic studies in yeast have implicated CAPs in vesicle trafficking and endocytosis. CAPs play a developmental role in multicellular organisms, and studies of Drosophila have illuminated the importance of the actin cytoskeleton during eye development and in establishing oocyte polarity. This review will highlight the critical structural and functional domains of CAPs, describe recent studies that have implied important roles for these proteins in linking cell signaling with actin polymerization, and highlight their roles in vesicle trafficking and development.

  1. Review of the neutrophil response to Bordetella pertussis infection.

    PubMed

    Eby, Joshua C; Hoffman, Casandra L; Gonyar, Laura A; Hewlett, Erik L

    2015-12-01

    The nature and timing of the neutrophil response to infection with Bordetella pertussis is influenced by multiple virulence factors expressed by the bacterium. After inoculation of the host airway, the recruitment of neutrophils signaled by B. pertussis lipooligosaccharide (LOS) is suppressed by pertussis toxin (PTX). Over the next week, the combined activities of PTX, LOS and adenylate cyclase toxin (ACT) result in production of cytokines that generate an IL-17 response, promoting neutrophil recruitment which peaks at 10-14 days after inoculation in mice. Arriving at the site of infection, neutrophils encounter the powerful local inhibitory activity of ACT, in conjunction with filamentous hemagglutinin. With the help of antibodies, neutrophils contribute to clearance of B. pertussis, but only after 28-35 days in a naïve mouse. Studies of the lasting, antigen-specific IL-17 response to infection in mice and baboons has led to progress in vaccine development and understanding of pathogenesis. Questions remain about the mediators that coordinate neutrophil recruitment and the mechanisms by which neutrophils overcome B. pertussis virulence factors.

  2. Review of the neutrophil response to Bordetella pertussis infection

    PubMed Central

    Eby, Joshua C.; Hoffman, Casandra L.; Gonyar, Laura A.; Hewlett, Erik L.

    2015-01-01

    The nature and timing of the neutrophil response to infection with Bordetella pertussis is influenced by multiple virulence factors expressed by the bacterium. After inoculation of the host airway, the recruitment of neutrophils signaled by B. pertussis lipooligosaccharide (LOS) is suppressed by pertussis toxin (PTX). Over the next week, the combined activities of PTX, LOS and adenylate cyclase toxin (ACT) result in production of cytokines that generate an IL-17 response, promoting neutrophil recruitment which peaks at 10–14 days after inoculation in mice. Arriving at the site of infection, neutrophils encounter the powerful local inhibitory activity of ACT, in conjunction with filamentous hemagglutinin. With the help of antibodies, neutrophils contribute to clearance of B. pertussis, but only after 28–35 days in a naïve mouse. Studies of the lasting, antigen-specific IL-17 response to infection in mice and baboons has led to progress in vaccine development and understanding of pathogenesis. Questions remain about the mediators that coordinate neutrophil recruitment and the mechanisms by which neutrophils overcome B. pertussis virulence factors. PMID:26432818

  3. Pituitary Adenlylate Cyclase Activating Peptide Protects Adult Neural Stem Cells from a Hypoglycaemic milieu

    PubMed Central

    Mansouri, Shiva; Lietzau, Grazyna; Lundberg, Mathias; Nathanson, David; Nyström, Thomas; Patrone, Cesare

    2016-01-01

    Hypoglycaemia is a common side-effect of glucose-lowering therapies for type-2 diabetic patients, which may cause cognitive/neurological impairment. Although the effects of hypoglycaemia in the brain have been extensively studied in neurons, how hypoglycaemia impacts the viability of adult neural stem cells (NSCs) has been poorly investigated. In addition, the cellular and molecular mechanisms of how hypoglycaemia regulates NSCs survival have not been characterized. Recent work others and us have shown that the pituitary adenylate cyclase-activating polypeptide (PACAP) and the glucagon-like peptide-1 receptor (GLP-1R) agonist Exendin-4 stimulate NSCs survival against glucolipoapoptosis. The aim of this study was to establish an in vitro system where to study the effects of hypoglycaemia on NSC survival. Furthermore, we determine the potential role of PACAP and Exendin-4 in counteracting the effect of hypoglycaemia. A hypoglycaemic in vitro milieu was mimicked by exposing subventricular zone-derived NSC to low levels of glucose. Moreover, we studied the potential involvement of apoptosis and endoplasmic reticulum stress by quantifying protein levels of Bcl-2, cleaved caspase-3 and mRNA levels of CHOP. We show that PACAP via PAC-1 receptor and PKA activation counteracts impaired NSC viability induced by hypoglycaemia. The protective effect induced by PACAP correlated with endoplasmic reticulum stress, Exendin-4 was ineffective. The results show that hypoglycaemia decreases NSC viability and that this effect can be substantially counteracted by PACAP via PAC-1 receptor activation. The data supports a potential therapeutic role of PAC-1 receptor agonists for the treatment of neurological complications, based on neurogenesis impairment by hypoglycaemia. PMID:27305000

  4. Novel hopanoid cyclases from the environment.

    PubMed

    Pearson, Ann; Flood Page, Sarah R; Jorgenson, Tyler L; Fischer, Woodward W; Higgins, Meytal B

    2007-09-01

    Hopanoids are ubiquitous isoprenoid lipids found in modern biota, in recent sediments and in low-maturity sedimentary rocks. Because these lipids primarily are derived from bacteria, they are used as proxies to help decipher geobiological communities. To date, much of the information about sources of hopanoids has come from surveys of culture collections, an approach that does not address the vast fraction of prokaryotic communities that remains uncharacterized. Here we investigated the phylogeny of hopanoid producers using culture-independent methods. We obtained 79 new sequences of squalene-hopene cyclase genes (sqhC) from marine and lacustrine bacterioplankton and analysed them along with all 31 sqhC fragments available from existing metagenomics libraries. The environmental sqhCs average only 60% translated amino acid identity to their closest relatives in public databases. The data imply that the sources of these important geologic biomarkers remain largely unknown. In particular, genes affiliated with known cyanobacterial sequences were not detected in the contemporary environments analysed here, yet the geologic record contains abundant hopanoids apparently of cyanobacterial origin. The data also suggest that hopanoid biosynthesis is uncommon: < 10% of bacterial species may be capable of producing hopanoids. A better understanding of the contemporary distribution of hopanoid biosynthesis may reveal fundamental insight about the function of these compounds, the organisms in which they are found, and the environmental signals preserved in the sedimentary record.

  5. Receptor Guanylyl Cyclases in Sensory Processing

    PubMed Central

    Maruyama, Ichiro N.

    2017-01-01

    Invertebrate models have generated many new insights into transmembrane signaling by cell-surface receptors. This review focuses on receptor guanylyl cyclases (rGCs) and describes recent advances in understanding their roles in sensory processing in the nematode, Caenorhabditis elegans. A complete analysis of the C. elegans genome elucidated 27 rGCs, an unusually large number compared with mammalian genomes, which encode 7 rGCs. Most C. elegans rGCs are expressed in sensory neurons and play roles in sensory processing, including gustation, thermosensation, olfaction, and phototransduction, among others. Recent studies have found that by producing a second messenger, guanosine 3′,5′-cyclic monophosphate, some rGCs act as direct sensor molecules for ions and temperatures, while others relay signals from G protein-coupled receptors. Interestingly, genetic and biochemical analyses of rGCs provide the first example of an obligate heterodimeric rGC. Based on recent structural studies of rGCs in mammals and other organisms, molecular mechanisms underlying activation of rGCs are also discussed in this review. PMID:28123378

  6. Control of Outflow Resistance by Soluble Adenylyl Cyclase

    PubMed Central

    Lee, Yong Suk

    2014-01-01

    Abstract Glaucoma is a leading cause of blindness in the United States affecting as many as 2.2 million Americans. All current glaucoma treatment strategies aim to reduce intraocular pressure, even in patients with normal tension glaucoma. Typically, this is accomplished by reducing the rate of aqueous flow by limiting aqueous production or enhancing drainage using drugs and surgery. Whereas these strategies are effective in diminishing vision loss, some patients continue to lose vision and many discontinue use of their medications because of undesirable side effects. Drugs known to be effective in altering conventional outflow have for the most part been abandoned from modern clinical practice due to undesirable side effects. Identification of new drugs that could enhance conventional outflow, would offer additional options in the treatment of glaucoma and ocular hypertension. To this end, our laboratory has recently uncovered a novel pathway for regulation of conventional outflow by the ciliary body. This pathway is dependent on soluble adenylyl cyclase, an enzyme that catalyzes the generation of cyclic adenosine 3′,5′ monophosphate (cAMP) in response to bicarbonate. PMID:24320087

  7. Structural evidence for variable oligomerization of the N-terminal domain of cyclase-associated protein (CAP).

    PubMed

    Yusof, Adlina Mohd; Hu, Nien-Jen; Wlodawer, Alexander; Hofmann, Andreas

    2005-02-01

    Cyclase-associated protein (CAP) is a highly conserved and widely distributed protein that links the nutritional response signaling to cytoskeleton remodeling. In yeast, CAP is a component of the adenylyl cyclase complex and helps to activate the Ras-mediated catalytic cycle of the cyclase. While the N-terminal domain of CAP (N-CAP) provides a binding site for adenylyl cyclase, the C-terminal domain (C-CAP) possesses actin binding activity. Our attempts to crystallize full-length recombinant CAP from Dictyostelium discoideum resulted in growth of orthorhombic crystals containing only the N-terminal domain (residues 42-227) due to auto-proteolytic cleavage. The structure was solved by molecular replacement with data at 2.2 A resolution. The present crystal structure allows the characterization of a head-to-tail N-CAP dimer in the asymmetric unit and a crystallographic side-to-side dimer. Comparison with previously published structures of N-CAP reveals variable modes of dimerization of this domain, but the presence of a common interface for the side-to-side dimer.

  8. Isolation of an Arabidopsis mutant lacking vitamin E and identification of a cyclase essential for all tocopherol biosynthesis.

    PubMed

    Porfirova, Svetlana; Bergmuller, Eveline; Tropf, Susanne; Lemke, Rainer; Dormann, Peter

    2002-09-17

    Tocopherol (vitamin E) is a plant chloroplast lipid presumed to be involved in the response to oxidative stress. A tocopherol-deficient mutant (vte1) was isolated from Arabidopsis thaliana by using a TLC-based screening approach. Mutant plants lacked all four tocopherol forms and were deficient in tocopherol cyclase activity. Genetic mapping of vte1 and a genomics-based approach led to the identification of the ORF At4g32770 as a candidate gene for tocopherol cyclase. In vte1, At4g32770 contains a splicing site mutation and the corresponding mRNA expression is reduced. Expression of VTE1 in Escherichia coli resulted in the production of a protein with high tocopherol cyclase and tocotrienol cyclase activity. The VTE1 sequence shows no similarities to genes with known function, but is similar to that of SXD1, a gene that was recently isolated based on the availability of the sucrose export defective1 maize mutant (sxd1). Growth of the vte1 mutant, chlorophyll content, and photosynthetic quantum yield were similar to wild type under optimal growth conditions. Therefore, absence of tocopherol has no large impact on photosynthesis or plant viability, suggesting that other antioxidants can compensate for the loss of tocopherol. During photo-oxidative stress, chlorophyll content and photosynthetic quantum yield were slightly reduced in vte1 as compared with wild type indicating a potential role for tocopherol in maintaining an optimal photosynthesis rate under high-light stress.

  9. Mutants of PC12 cells with altered cyclic AMP responses

    SciTech Connect

    Block, T.; Kon, C.; Breckenridge, B.M.

    1984-10-01

    PCl2 cells, derived from a rat pheochromocytoma, were mutagenized and selected in media containing agents known to elevate intracellular concentrations of cyclic AMP (cAMP). More than 40 clones were isolated by selection with cholera toxin or 2-chloroadenosine or both. The variants that were deficient in accumulating cAMP were obtained by using a protocol in which 1 ..mu..m 8-bromo-cAMP was included in addition to the agonist. Certain of these variants were partially characterized with respect to the site of altered cAMP metabolism. The profiles of adenylate cyclase activity responsiveness of certain variants to guanosine-5'-(BETA,..gamma..-imido) triphosphate and to forskolin resembled those of UNC and cyc phenotypes of S49 lymphoma cells, which are functionally deficient in the GTP-sensitive coupling protein, N/sub s/. Other variants were characterized by increased cyclic nucleotide phosphodiesterase activity at low substrate concentration. Diverse morphological traits were observed among the variants, but it was not possible to assign them to a particular cAMP phenotype. Two revertants of a PCl2 mutant were isolated and observed to have regained a cellular cAMP response to 2-chloroadenosine and to forskolin. It is hoped that these PCl2 mutants will have utility for defining cAMP-mediated functions, including any links to the action of nerve growth factor, in cells derived from the neural crest.

  10. Role of glutaminyl cyclases in thyroid carcinomas.

    PubMed

    Kehlen, Astrid; Haegele, Monique; Menge, Katja; Gans, Kathrin; Immel, Uta-Dorothee; Hoang-Vu, Cuong; Klonisch, Thomas; Demuth, Hans-Ulrich

    2013-02-01

    CCL2 is a chemokine known to recruit monocytes/macrophages to sites of inflammation. CCL2 is also associated with tumor progression in several cancer types. Recently, we showed that the N-terminus of CCL2 is modified to a pyroglutamate (pE)-residue by both glutaminyl cyclases (QC (QPCT)) and its isoenzyme (isoQC (QPCTL)). The pE-residue increases stability against N-terminal degradation by aminopeptidases. Here, we report an upregulation of QPCT expression in tissues of patients with thyroid carcinomas compared with goiter tissues, whereas QPCTL was not regulated. In thyroid carcinoma cell lines, QPCT gene expression correlates with the mRNA levels of its substrate CCL2. Both QPCT and CCL2 are regulated in a NF-κB-dependent pathway shown by stimulation with TNFa and IL1b as well as by inhibition with the IKK2 inhibitor and RNAi of p50. In the culture supernatant of thyroid carcinoma cells, equal amounts of pECCL2 and total CCL2 were detected by two ELISAs discriminating between total CCL2 and pECCL2, concluding that all CCL2 is secreted as pECCL2. Activation of the CCL2/CCR2 pathway by recombinant CCL2 increased tumor cell migration of FTC238 cells in scratch assays as well as thyroid carcinoma cell-derived CCL2-induced migration of monocytic THP1 cells. Suppression of CCL2 signaling by CCR2 antagonist, IKK2 inhibitor, and QPCT RNAi reduced FTC238 cell growth measured by WST8 proliferation assays. Our results reveal new evidence for a novel role of QC in thyroid carcinomas and provide an intriguing rationale for the use of QC inhibitors as a means of blocking pECCL2 formation and preventing thyroid cancer metastasis.

  11. 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.

  12. Mouse hypothalamic growth hormone-releasing hormone and somatostatin responses to probes of signal transduction systems.

    PubMed

    Sato, M; Downs, T R; Frohman, L A

    1993-01-01

    Signal transduction mechanisms involved in mouse growth hormone-releasing hormone (GRH) and somatostatin (SRIH) release were investigated using an in vitro perifusion system. Hypothalamic fragments were exposed to depolarizing agents, protein kinase A and C activators, and a calcium ionophore. The depolarizing agents, KCl (60 mM) and veratridine (50 microM), induced similar patterns of GRH and SRIH release. Somatostatin release in response to both agents was twofold greater than that of GRH. Forskolin (10 microM and 100 microM), an adenylate cyclase activator, stimulated both GRH and SRIH release, though with different secretory profiles. The SRIH response was prolonged and persisted beyond removal of the drug from the system, while the GRH response was brief, ending even prior to forskolin removal. Neither GRH nor SRIH were stimulated by 1,9-dideoxy-forskolin (100 microM), a forskolin analog with cAMP-independent actions. A23187 (5 microM), a calcium ionophore, stimulated the release of SRIH to a much greater extent than that of GRH. The GRH and SRIH secretory responses to PMA (1 microM), a protein kinase C activator, were similar, though delayed. The results suggest that 1) GRH and SRIH secretion are regulated by both protein kinase A and C pathways, and 2) depolarizing agents are important for the release of both hormones.

  13. Regulation of cGMP levels by guanylate cyclase in truncated frog rod outer segments

    PubMed Central

    1989-01-01

    Cyclic GMP is the second messenger in phototransduction and regulates the photoreceptor current. In the present work, we tried to understand the regulation mechanism of cytoplasmic cGMP levels in frog photoreceptors by measuring the photoreceptor current using a truncated rod outer segment (tROS) preparation. Since exogenously applied substance diffuses into tROS from the truncated end, we could examine the biochemical reactions relating to the cGMP metabolism by manipulating the cytoplasmic chemical condition. In tROS, exogenously applied GTP produced a dark current whose amplitude was half-maximal at approximately 0.4 mM GTP. The conductance for this current was suppressed by light in a fashion similar to when it is activated by cGMP. In addition, no current was produced in the absence of Mg2+, which is known to be necessary for the guanylate cyclase activity. These results indicate that guanylate cyclase was present in tROS and synthesized cGMP from exogenously applied GTP. The enzyme activity was distributed throughout the rod outer segment. The amount of synthesized cGMP increased as the cytoplasmic Ca2+ concentration of tROS decreased, which indicated the activation of guanylate cyclase at low Ca2+ concentrations. Half-maximal effect of Ca2+ was observed at approximately 100 nM. tROS contained the proteins involved in the phototransduction mechanism and therefore, we could examine the regulation of the light response waveform by Ca2+. At low Ca2+ concentrations, the time course of the light response was speeded up probably because cGMP recovery was facilitated by activation of the cyclase. Then, if the cytoplasmic Ca2+ concentration of a photoreceptor decreases during light stimulation, the Ca2+ decrease may explain the acceleration of the light response during light adaptation. In tROS, however, we did observe an acceleration during repetitive light flashes when the cytoplasmic Ca2+ concentration increased during the stimulation. This result suggests the

  14. Chirally selective, intramolecular interaction observed in an aminoacyl adenylate anhydride

    NASA Technical Reports Server (NTRS)

    Lacey, J. C., Jr.; Hall, L. M.; Mullins, D. W., Jr.; Watkins, C. L.

    1985-01-01

    The interaction between amino acids and nucleotide bases is studied. The proton NMR spectrum of N-acetylphenylalanyl-AMP-anhydride is analyzed H8 and H2 signals, two upfield signals of equal size, and five phenylalanine ring proton signals are observed in the spectrum; the upfield movement of the proton and the racemization of the N-acetyl L-phenylalanine material are examined. The differences in the position of the signals due to the diastereoisomers are investigated. The separation of the D and L amino acyl adenylates using HPLC is described. H-1 NMR spectra of the isomers are examined in order to determine which isomer displays the strongest interaction between the phenyl ring and the adenine ring. The spectra reveal that the L isomer shows the highest upfield change of both H8 and H2 signals. It is noted that the phenyl ring lies over C2 of the adenine ring with the phenyl meta and para protons extended past the adenine ring and the phenyl ortho protons.

  15. Hydrolytic properties of phenylalanyl- and N-acetylphenylalanyl adenylate anhydrides

    NASA Technical Reports Server (NTRS)

    Lacey, J. C., Jr.; Mullins, D. W., Jr.; Senaratne, N.

    1984-01-01

    The hydrolysis of phenylalynyl- and N-acetylephenylalanyl adenylate anhydrides (AcPhe-AMP) is studied experimentally using a new spectrophotometric method. The hydrolysis process was analyzed at low concentrations (0.0001 M), constant temperature of 25 C, constant buffer concentration (0.05 M), and as a function of pH. It is found that while Phe-AMP is susceptible to attack by OH(-), AcPhe-AMP is susceptible to acid decomposition as well. At a pH of 4 to 8, Phe-AMP hydolyzes faster than AcPhe-AMP, but at pH less than four or greater than eight, the blocked form hydrolyzes faster. Both forms are attacked by H2O at the same rate. The rate laws for the various hydrolytic mechanisms and the activation energies for the hydrolyses at pH 7.1 are given in a table, and the possible relevance of the findings to the origin and evolution of the process of protein synthesis is discussed.

  16. Kinetics of inhibition of firefly luciferase by oxyluciferin and dehydroluciferyl-adenylate.

    PubMed

    Ribeiro, César; Esteves da Silva, Joaquim C G

    2008-09-01

    The inhibition mechanisms of the firefly luciferase (Luc) by the two major products of the reactions catalysed by Luc, oxyluciferin and dehydroluciferyl-adenylate (L-AMP), were investigated. Light production in the presence and absence of these inhibitors (0.5 to 2 microM oxyluciferin; 0.0025 to 1.25 microM L-AMP) has been measured in 50 mM Hepes buffer (pH=7.5), 10 nM Luc, 250 microM ATP and D-Luciferin (from 3.75 up to 120 microM). Nonlinear regression analysis with the appropriate kinetic models (Henri-Michaelis-Menten and William-Morrison equations) reveals that oxyluciferin is a competitive inhibitor of luciferase (Ki=0.50+/-0.03 microM) while L-AMP act as a tight-binding competitive inhibitor (Ki=3.8+/-0.7 nM). The Km values obtained both for oxyluciferin and L-AMP were 14.7+/-0.7 and 14.9+/-0.2 microM, respectively. L-AMP is a stronger inhibitor of Luc than oxyluciferin and the major responsible for the characteristic flash profile of in vitro Luc bioluminescence.

  17. Regulation and organization of adenylyl cyclases and cAMP.

    PubMed Central

    Cooper, Dermot M F

    2003-01-01

    Adenylyl cyclases are a critically important family of multiply regulated signalling molecules. Their susceptibility to many modes of regulation allows them to integrate the activities of a variety of signalling pathways. However, this property brings with it the problem of imparting specificity and discrimination. Recent studies are revealing the range of strategies utilized by the cyclases to solve this problem. Microdomains are a consequence of these solutions, in which cAMP dynamics may differ from the broad cytosol. Currently evolving methodologies are beginning to reveal cAMP fluctuations in these various compartments. PMID:12940771

  18. Intracellular cAMP signaling by soluble adenylyl cyclase

    PubMed Central

    Tresguerres, Martin; Levin, Lonny R.; Buck, Jochen

    2011-01-01

    Soluble adenylyl cyclase (sAC) is a recently identified source of the ubiquitous second messenger cAMP. sAC is distinct from the more widely studied source of cAMP, the transmembrane adenylyl cyclases (tmACs); its activity is uniquely regulated by bicarbonate anions, and it is distributed throughout the cytoplasm and in cellular organelles. Due to its unique localization and regulation, sAC has various functions in a variety of physiological systems which are distinct from tmACs. In this review, we detail the known functions of sAC, and we reassess commonly held views of cAMP signaling inside cells. PMID:21490586

  19. Oligomeric state affects oxygen dissociation and diguanylate cyclase activity of globin coupled sensors.

    PubMed

    Burns, Justin L; Deer, D Douglas; Weinert, Emily E

    2014-11-01

    Bacterial biofilm formation is regulated by enzymes, such as diguanylate cyclases, that respond to environmental signals and alter c-di-GMP levels. Diguanylate cyclase activity of two globin coupled sensors is shown to be regulated by gaseous ligands, with cyclase activity and O2 dissociation affected by protein oligomeric state.

  20. Studies on responsiveness of hepatoma cells to catecholamines. II. Comparison of beta-adrenergic responsiveness of rat ascites hepatoma cells with cultured normal rat liver cells.

    PubMed

    Miyamoto, K; Matsunaga, T; Takemoto, N; Sanae, F; Koshiura, R

    1985-05-01

    The pharmacological properties of beta-adrenoceptors in rat ascites hepatoma cells were compared with those in normal rat liver cells which were cultured for 24 hr after collagenase digestion. Adenylate cyclases in the homogenates of cultured normal rat liver cells and rat ascites hepatoma cells, AH44, AH66, AH109A, AH130 and AH7974, were all activated by isoproterenol or NaF to different degrees. The enzyme in rat liver cells was activated by several beta 2-agonists but those in all hepatoma cells hardly responded. Furthermore, salbutamol, a beta 2-partial agonist, antagonized the cyclase activation by isoproterenol in AH130 cells. The Kact value of isoproterenol for the activation of adenylate cyclase in AH130 cells was smaller than that in rat liver cells. A comparison of the Ki values of beta-antagonists for the inhibition of isoproterenol-stimulated cyclase activity shows that while the Ki values of propranolol and butoxamine in AH130 cells were similar to those in rat liver cells, a significant difference was observed in the values for beta 1-selective antagonists between AH130 cells and rat liver cells. The Ki values of metoprolol and atenolol for AH130 cells were 137- and 90-fold lower, respectively, than for normal rat liver cells. From these findings, it is strongly suggested that beta-adrenoceptors in rat ascites hepatoma cells including AH130 cells have similar properties to the mammalian beta 1-receptor.

  1. Glucagon receptor of human liver. Studies of its molecular weight and binding properties, and its ability to activate hepatic adenylyl cyclase of non-obese and obese subjects.

    PubMed Central

    Livingston, J N; Einarsson, K; Backman, L; Ewerth, S; Arner, P

    1985-01-01

    The glucagon receptor and the adenylyl cyclase system of human liver membranes were studied in six non-obese and six obese subjects who had elevated insulin and plasma glucagon levels. Analysis of specific glucagon binding by the method of Scatchard demonstrated a linear (monocomponent) plot with a dissociation constant of 2-3 nM, and the binding at low hormone concentrations was sensitive to guanosine triphosphate (GTP). The molecular weight of the glucagon receptor was 63,000 D as determined by an affinity labeling procedure and sodium dodecyl sulfate gel electrophoresis. Affinity labeling of this structure was specific for glucagon and inhibited by GTP. Glucagon stimulated the production of cyclic adenosine monophosphate (cAMP) by human membranes with half-maximal activation elicited by 6 nM hormone. The human cyclase system required GTP to facilitate an optimal glucagon response. NaF (10 mM) also activated the cyclase system and produced the same magnitude of response as maximum glucagon activation. A comparison of the liver adenylyl cyclase system of non-obese and obese subjects was made using glucagon (5 nM and 1 microM) and NaF (10 mM). No significant differences in cAMP production were noted between the two groups, regardless of the agent used to activate the enzyme. These findings agree with the glucagon binding studies that showed similar amounts of binding activity in the membranes from the two groups. Also, there was no influence of either age or sex of the subjects on the adenylyl cyclase response. In conclusion, human liver membranes contain a glucagon receptor and an adenylyl cyclase system that correspond closely to the well-studied system in animal liver. This system in human obesity is not altered by the approximately twofold elevation in plasma glucagon that occurs in this metabolic disorder. Images PMID:2982913

  2. Marine Bivalve Cellular Responses to Beta Blocker Exposures ...

    EPA Pesticide Factsheets

    β blockers are prescription drugs used for medical treatment of hypertension and arrhythmias. They prevent binding of agonists such as catecholamines to β adrenoceptors. In the absence of agonist induced activation of the receptor, adenylate cyclase is not activated which in turn limits cAMP production and protein kinase A activation, preventing increases in blood pressure and arrhythmias. After being taken therapeutically, commonly prescribed β blockers may make their way to coastal habitats via discharge from waste water treatment plants (WWTP) posing a potential risk to aquatic organisms. The aim of our research is to evaluate cellular responses of three commercially important marine bivalves - Eastern oysters, blue mussels and hard clams - upon exposure to two β blocker drugs, propranolol and metoprolol, and to find molecular initiating events (MIEs) indicative of the exposure. Bivalves were obtained from Narragansett Bay (Rhode Island, USA) and acclimated in the laboratory. Following acclimation, gills and hepatopancreas (HP) tissues were harvested and separately exposed to 0, 1, 10, 100 and 1000 ng/l of each drug. Tissues were bathed in 30 parts per thousand (ppt) filtered seawater, antibiotic mix, Leibovitz nutrient media, and the test drug. Exposures were conducted for 24 hours and samples were saved for cellular biomarker assays. A lysosomal destabilization assay, which is a marker of membrane damage, was also performed at the end of each exposure.

  3. Role of guanylate cyclase-activating proteins (GCAPs) in setting the flash sensitivity of rod photoreceptors

    PubMed Central

    Mendez, Ana; Burns, Marie E.; Sokal, Izabela; Dizhoor, Alexander M.; Baehr, Wolfgang; Palczewski, Krzysztof; Baylor, Denis A.; Chen, Jeannie

    2001-01-01

    The retina's photoreceptor cells adjust their sensitivity to allow photons to be transduced over a wide range of light intensities. One mechanism thought to participate in sensitivity adjustments is Ca2+ regulation of guanylate cyclase (GC) by guanylate cyclase-activating proteins (GCAPs). We evaluated the contribution of GCAPs to sensitivity regulation in rods by disrupting their expression in transgenic mice. The GC activity from GCAPs−/− retinas showed no Ca2+ dependence, indicating that Ca2+ regulation of GCs had indeed been abolished. Flash responses from dark-adapted GCAPs−/− rods were larger and slower than responses from wild-type rods. In addition, the incremental flash sensitivity of GCAPs−/− rods failed to be maintained at wild-type levels in bright steady light. GCAP2 expressed in GCAPs−/− rods restored maximal light-induced GC activity but did not restore normal flash response kinetics. We conclude that GCAPs strongly regulate GC activity in mouse rods, decreasing the flash sensitivity in darkness and increasing the incremental flash sensitivity in bright steady light, thereby extending the rod's operating range. PMID:11493703

  4. 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.

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

    PubMed Central

    Krumenacker, Joshua S.; Hyder, Salman M.; Murad, Ferid

    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 17β-estradiol (E2) regulates the α1 and β1 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 α1 10% and sGC β1 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. PMID:11209068

  6. Structural characterization of Burkholderia pseudomallei adenylate kinase (Adk): Profound asymmetry in the crystal structure of the ‘open’ state

    PubMed Central

    Buchko, Garry W.; Robinson, Howard; Abendroth, Jan; Staker, Bart L.; Myler, Peter J.

    2010-01-01

    In all organisms adenylate kinases (Adks) play a vital role in cellular energy metabolism and nucleic acid synthesis. Due to differences in catalytic properties between the Adks found in prokaryotes and in the cytoplasm of eukaryotes, there is interest in targeting this enzyme for new drugs therapies against infectious bacterial agents. Here we report the 2.1 Å resolution crystal structure for the 220-residue Adk from Burkholderia pseudomallei (BpAdk), the etiological agent responsible for the infectious disease meliodosis. The general structure of apo BpAdk is similar to other Adk structures, composed of a CORE subdomain with peripheral ATP-binding (ATPbd) and LID subdomains. The two molecules in the asymmetric unit have significantly different conformations, with a backbone RMSD of 1.46 Å. These two BpAdk conformations may represent ‘open’ Adk sub-states along the preferential pathway to the ‘closed’ substrate-bound state. PMID:20331978

  7. Structural characterization of Burkholderia pseudomallei adenylate kinase (Adk): Profound asymmetry in the crystal structure of the 'open' state

    SciTech Connect

    Buchko, G.W.; Robinson, H.; Abendroth, J.; Staker, B. L.; Myler, P. J.

    2010-04-16

    In all organisms adenylate kinases (Adks) play a vital role in cellular energy metabolism and nucleic acid synthesis. Due to differences in catalytic properties between the Adks found in prokaryotes and in the cytoplasm of eukaryotes, there is interest in targeting this enzyme for new drug therapies against infectious bacterial agents. Here we report the 2.1 {angstrom} resolution crystal structure for the 220-residue Adk from Burkholderia pseudomallei (BpAdk), the etiological agent responsible for the infectious disease melioidosis. The general structure of apo BpAdk is similar to other Adk structures, composed of a CORE subdomain with peripheral ATP-binding (ATP{sub bd}) and LID subdomains. The two molecules in the asymmetric unit have significantly different conformations, with a backbone RMSD of 1.46 {angstrom}. These two BpAdk conformations may represent 'open' Adk sub-states along the preferential pathway to the 'closed' substrate-bound state.

  8. Photoactivation mechanism of a bacterial light-regulated adenylyl cyclase.

    PubMed

    Lindner, Robert; Hartmann, Elisabeth; Tarnawski, Miroslaw; Winkler, Andreas; Frey, Daniel; Reinstein, Jochen; Meinhart, Anton; Schlichting, Ilme

    2017-03-20

    Light-regulated enzymes enable organisms to quickly respond to changing light conditions. We characterize a photoactivatable adenylyl cyclase from Beggiatoa sp. (bPAC) that translates a blue light signal into production of the second messenger cyclic AMP. bPAC contains a BLUF photoreceptor domain that senses blue light using a flavin chromophore, linked to an adenylyl cyclase (AC) domain. We present a dark state crystal structure of bPAC that closely resembles the recently published structure of the homologous OaPAC from Oscillatoria acuminata. To elucidate the structural mechanism of light-dependent AC activation by the BLUF domain, we determined crystal structures of illuminated bPAC and of a pseudo-lit state variant. We use hydrogen-deuterium exchange measurements of secondary structure dynamics and hypothesis-driven point mutations to trace the activation pathway from the chromophore in the BLUF domain to the active site of the cyclase. The structural changes are relayed from the residues interacting with the excited chromophore through a conserved kink of the BLUF β-sheet to a tongue-like extrusion of the AC domain that regulates active site opening and repositions catalytic residues. Our findings not only show the specific molecular pathway of photoactivation in BLUF-regulated ACs, but they also have implications for the general understanding of signaling in BLUF domains and of the activation of adenylyl cyclases.

  9. Synthesis of amino acyl adenylates using the tert-butoxycarbonyl protecting group

    NASA Technical Reports Server (NTRS)

    Armstrong, D. W.; Seguin, R.; Saburi, M.; Fendler, J. H.

    1979-01-01

    The synthesis of amino acyl adenylates using N-tert-butoxycarbonyl-protected amino acids is reported. Anhydrous solutions containing N-tert-butoxycarbonyl alanine, phenylalanine, and methionine were combined with the anhydrous mono (tri-n-octylammonium) salt of adenosine 5'-phosphate and the resultant amino acyl adenylates were characterized by means of elemental analysis, and infrared and proton NMR spectroscopy. Amino acyl adenylate yields of up to 60% were obtained with high purity at room temperatures. The reported synthesis is considered to represent a large improvement over previous methods due to the purity of the products, normal temperature requirements, and the stability of the starting compounds, which suggests its use in investigations of prebiotic oligo- and polypeptide synthesis.

  10. A Survey of Nucleotide Cyclases in Actinobacteria: Unique Domain Organization and Expansion of the Class III Cyclase Family in Mycobacterium tuberculosis

    PubMed Central

    Sivakumar, K.; Krupa, A.; Srinivasan, N.

    2004-01-01

    Cyclic nucleotides are well-known second messengers involved in the regulation of important metabolic pathways or virulence factors. There are six different classes of nucleotide cyclases that can accomplish the task of generating cAMP, and four of these are restricted to the prokaryotes. The role of cAMP has been implicated in the virulence and regulation of secondary metabolites in the phylum Actinobacteria, which contains important pathogens, such as Mycobacterium tuberculosis, M. leprae, M. bovis and Corynebacterium, and industrial organisms from the genus Streptomyces. We have analysed the actinobacterial genome sequences found in current databases for the presence of different classes of nucleotide cyclases, and find that only class III cyclases are present in these organisms. Importantly, prominent members such as M. tuberculosis and M. leprae have 17 and 4 class III cyclases, respectively, encoded in their genomes, some of which display interesting domain fusions seen for the first time. In addition, a pseudogene corresponding to a cyclase from M. avium has been identified as the only cyclase pseudogene in M. tuberculosis and M. bovis. The Corynebacterium and Streptomyces genomes encode only a single adenylyl cyclase each, both of which have corresponding orthologues in M. tuberculosis. A clustering of the cyclase domains in Actinobacteria reveals the presence of typical eukaryote-like, fungi-like and other bacteria-like class III cyclase sequences within this phylum, suggesting that these proteins may have significant roles to play in this important group of organisms. PMID:18629044

  11. A subnanomolar concentration of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) pre-synaptically modulates glutamatergic transmission in the rat hippocampus acting through acetylcholine.

    PubMed

    Pecoraro, Valeria; Sardone, Lara Maria; Chisari, Mariangela; Licata, Flora; Li Volsi, Guido; Perciavalle, Vincenzo; Ciranna, Lucia; Costa, Lara

    2017-01-06

    The neuropeptide PACAP modulates synaptic transmission in the hippocampus exerting multiple effects through different receptor subtypes: the underlying mechanisms have not yet been completely elucidated. The neurotransmitter acetylcholine (ACh) also exerts a well-documented modulation of hippocampal synaptic transmission and plasticity. Since PACAP was shown to stimulate ACh release in the hippocampus, we tested whether PACAP acting through ACh might indirectly modulate glutamate-mediated synaptic transmission at a pre- and/or at a post-synaptic level. Using patch clamp on rat hippocampal slices, we tested PACAP effects on stimulation-evoked AMPA receptor-mediated excitatory post-synaptic currents (EPSCsAMPA) in the CA3-CA1 synapse and on spontaneous miniature EPSCs (mEPSCs) in CA1 pyramidal neurons. A subnanomolar dose of PACAP (0.5nM) decreased EPSCsAMPA amplitude, enhanced EPSC paired-pulse facilitation (PPF) and reduced mEPSC frequency, indicating a pre-synaptic decrease of glutamate release probability: these effects were abolished by simultaneous blockade of muscarinic and nicotinic ACh receptors, indicating the involvement of endogenous ACh. The effect of subnanomolar PACAP was abolished by a PAC1 receptor antagonist but not by a VPAC receptor blocker. At a higher concentration (10nM), PACAP inhibited EPSCsAMPA: this effect persisted in the presence of ACh receptor antagonists and did not involve any change in PPF or in mEPSC frequency, thus was not mediated by ACh and was exerted post- synaptically on CA1 pyramidal neurons. We suggest that a high-affinity PAC1 receptor pre-synaptically modulates hippocampal glutamatergic transmission acting through ACh. Therefore, administration of PACAP at very low doses might be envisaged in cognitive diseases with reduced cholinergic transmission.

  12. Rapid molecular pathotyping of major salmonella enterica serotypes based on single-nucleotide polymorphisms (SNPs) in the adenylate cyclase (cyaA) gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Introduction: Salmonella enterica subsp. enterica serotype Enteriditis (S. Enteriditis) is the leading cause of salmonellosis worldwide, including the USA. Many S. enterica serotypes known to cause foodborne disease are associated with broiler meat contamination. While some serotypes are specific...

  13. H3 receptor-mediated inhibition of noradrenaline release: an investigation into the involvement of Ca2+ and K+ ions, G protein and adenylate cyclase.

    PubMed

    Schlicker, E; Kathmann, M; Detzner, M; Exner, H J; Göthert, M

    1994-07-01

    The present study was aimed at the identification of mechanisms following the activation of histamine H3 receptors. Mouse brain cortex slices preincubated with 3H-noradrenaline were superfused and the (H3 receptor-mediated) effect of histamine on the electrically evoked tritium overflow was studied under a variety of conditions. The extent of inhibition produced by histamine was inversely related to the frequency of stimulation used to evoke tritium overflow and to the Ca2+ concentration in the superfusion medium. An activator (levcromakalim) and blocker (glibenclamide) of ATP-dependent K+ channels did not affect the electrically evoked tritium overflow and its inhibition by histamine. A blocker of voltage-sensitive K+ channels, tetraethylammonium (TEA), increased the evoked overflow and attenuated the inhibitory effect of histamine. TEA also reduced the inhibitory effect of noradrenaline and prostaglandin E2 on the evoked overflow. When the facilitatory effect of TEA on the evoked overflow was compensated for by reducing the Ca2+ concentration in the superfusion medium, TEA did no longer attenuate the effect of histamine. Exposure of the slices to the SH group-alkylating agent N-ethylmaleimide increased the evoked overflow and attenuated the inhibitory effect of histamine; both effects were counteracted by the SH group-protecting agent dithiothreitol, which, by itself, did not affect the evoked overflow and its inhibition by histamine. Mouse brain cortex membranes were used to study the effect of the H3 receptor agonist R-(-)-alpha-methylhistamine on the basal cAMP accumulation and on the accumulation stimulated by forskolin or noradrenaline.(ABSTRACT TRUNCATED AT 250 WORDS)

  14. Bicarbonate-sensitive soluble and transmembrane adenylyl cyclases in peripheral chemoreceptors

    PubMed Central

    Nunes, Ana R.; Holmes, Andrew P.S.; Sample, Vedangi; Kumar, Prem; Cann, Martin J.; Monteiro, Emília C.; Zhang, Jin; Gauda, Estelle B.

    2014-01-01

    Stimulation of the carotid body (CB) chemoreceptors by hypercapnia triggers a reflex ventilatory response via a cascade of cellular events, which includes generation of cAMP. However, it is not known if molecular CO2/HCO3− and/or H+ mediate this effect and how these molecules contribute to cAMP production. We previously reported that the CB highly expresses HCO3−-sensitive soluble adenylyl cyclase (sAC). In the present study we systematically characterize the role of sAC in the CB, comparing the effect of isohydric hypercapnia (IH) in cAMP generation through activation of sAC or transmembrane-adenylyl cyclase (tmAC). Pharmacological deactivation of sAC and tmAC decreased the CB cAMP content in normocapnia and IH with no differences between these two conditions. Changes from normocapnia to IH did not effect the degree of PKA activation and the carotid sinus nerve discharge frequency. sAC and tmAC are functional in CB but intracellular elevations in CO2/HCO3− in IH conditions on their own are insufficient to further activate these enzymes, suggesting that the hypercapnic response is dependent on secondary acidosis. PMID:23727159

  15. Clay catalyzed polymerization of amino acid adenylates and its relationship to biochemical reactions

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, M.

    1978-01-01

    The adsorption and polymerization of alanine adenylate on montmorillonite at pH 7 when either its interspacial faces or its edger are blocked by an excess of histidine or sodium hexametaphosphate was investigated. Results indicate that alanine adenylate can be adsorbed any place on the interspacial spaces of the clay; however, adsorption of its phosphate part, which is limited to the edges of the clay, is necessary for polymerization to occur. As a result, polymerization takes place only at sites on the interspacial faces bordering the edges.

  16. The polymerization of amino acid adenylates on sodium-montmorillonite with preadsorbed polypeptides

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, Mella; Eirich, Frederick R.

    1988-01-01

    The spontaneous polymerization of amino acid adenylates on Na-montmorillonite in dilute, neutral suspension, after polypeptides were adsorbed on the clay, is studied. It is found that the degrees of polymerization of the oligopeptides and polypeptides obtained is dependent on the amounts of polypeptides that were preadsorbed. It is concluded that a catalytic activity may derive from c-spacings that offer adsorption sites for the reagent amino acid adenylate within the peripheral recesses of irregularly stacked clay platelets by bringing the anhydride bonds and neutral amino groups into favorable reaction distances.

  17. Structure of the N-terminal domain of the adenylyl cyclase-associated protein (CAP) from Dictyostelium discoideum.

    PubMed

    Ksiazek, Dorota; Brandstetter, Hans; Israel, Lars; Bourenkov, Gleb P; Katchalova, Galina; Janssen, Klaus-Peter; Bartunik, Hans D; Noegel, Angelika A; Schleicher, Michael; Holak, Tad A

    2003-09-01

    Cyclase-associated proteins (CAPs) are widely distributed and highly conserved proteins that regulate actin remodeling in response to cellular signals. The N termini of CAPs play a role in Ras signaling and bind adenylyl cyclase; the C termini bind to G-actin and thereby alter the dynamic rearrangements of the microfilament system. We report here the X-ray structure of the core of the N-terminal domain of the CAP from Dictyostelium discoideum, which comprises residues 51-226, determined by a combination of single isomorphous replacement with anomalous scattering (SIRAS). The overall structure of this fragment is an alpha helix bundle composed of six antiparallel helices. Results from gel filtration and crosslinking experiments for CAP(1-226), CAP(255-464), and the full-length protein, together with the CAP N-terminal domain structure and the recently determined CAP C-terminal domain structure, provide evidence that the functional structure of CAP is multimeric.

  18. Epoxygenase metabolites of arachidonic acid inhibit vasopressin response in toad bladder

    SciTech Connect

    Schlondorff, D.; Petty, E.; Oates, J.A.; Jacoby, M.; Levine, S.D. Vanderbilt Univ., Nashville, TN )

    1987-09-01

    In addition to cyclooxygenase and lipoxygenase pathways, the kidney can also metabolize arachidonic acid by a NADPH-dependent cytochrome P-450 enzyme to epoxyeicosatrienoic acids (EETs); furthermore, 5,6-EET has been shown to alter electrolyte transport across isolated renal tubules. The authors examined the effects of three ({sup 14}C-labeled)-EETs (5,6-, 11,12-, and 14,15-EET) on osmotic water flow across toad urinary bladder. All three EETs reversibly inhibited vasopressin-stimulated osmotic water flow with 5,6- and 11,12-EET being the most potent. The effects appeared to be independent of prostaglandins EETs inhibited the water flow response to forskolin but not the response to adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) or 8-BrcAMP, consistent with an effect on cAMP generation. To determine whether these effects were due to the EETs or to products of their metabolism, they examined the effects of their vicinal diol hydrolysis products, the dihydroxyeicosatrienoic acids. Nonenzymatic conversion of labeled 5,6-EET to its vicinal diol occurred rapidly in the buffer, whereas 11,12-EET was hydrolyzed in a saturable manner only when incubated in the presence of bladder tissue. The dihydroxyeicosatrienoic acids formed inhibited water flow in a manner paralleling that of the EETs. The data support the hypothesis that EETs and their physiologically active dihydroxyeicosatrienoic acid metabolites inhibit vasopressin-stimulated water flow predominantly via inhibition of adenylate cyclase.

  19. Effects of electromagnetic stimuli on bone and bone cells in vitro: inhibition of responses to parathyroid hormone by low-energy low-frequency fields.

    PubMed

    Luben, R A; Cain, C D; Chen, M C; Rosen, D M; Adey, W R

    1982-07-01

    Low-energy electromagnetic fields pulsed at frequencies of 10-90 Hz significantly increase healing of chronic fracture nonunions in man. These fields are effective at tissue current levels several orders of magnitude lower than those required for transmembrane depolarization of normal cells. We have examined the effects of two clinically used pulsed electromagnetic fields on cultures of the osteoblast-like mouse bone cell line MMB-1. Both fields significantly reduced cellular production of cAMP in response to parathyroid hormone and osteoclast activating factor. Neither basal nor fluoride-activated levels of adenylate cyclase were altered in membranes from cells cultured in the fields; however, the same membrane preparations exhibited markedly inhibited responses to parathyroid hormone. The fields blocked the inhibitory effects of the hormone on collagen synthesis by MMB-1 cells. However, there was no effect on the inhibition of collagen synthesis by 1,25-dihydroxyvitamin D(3), which is believed to act primarily by a nuclear, rather than by a membrane-dependent, mechanism. No significant differences were noted between effects of the two fields, one generating continuous pulse trains (72 Hz) and the other generating recurrent bursts (15 Hz) of shorter pulses. We hypothesize that these field effects are mediated primarily at the plasma membrane of osteoblasts, either by interference with hormone-receptor interactions or by blocking of receptor-cyclase coupling in the membrane. These responses occurred with induced extracellular fields of 1 mV/cm or less, even though transmembrane potential gradients are typically 10(5) V/cm.

  20. Effects of electromagnetic stimuli on bone and bone cells in vitro: Inhibition of responses to parathyroid hormone by low-energy low-frequency fields

    PubMed Central

    Luben, Richard A.; Cain, Christopher D.; Chen, Monica Chi-Yun; Rosen, David M.; Adey, W. Ross

    1982-01-01

    Low-energy electromagnetic fields pulsed at frequencies of 10-90 Hz significantly increase healing of chronic fracture nonunions in man. These fields are effective at tissue current levels several orders of magnitude lower than those required for transmembrane depolarization of normal cells. We have examined the effects of two clinically used pulsed electromagnetic fields on cultures of the osteoblast-like mouse bone cell line MMB-1. Both fields significantly reduced cellular production of cAMP in response to parathyroid hormone and osteoclast activating factor. Neither basal nor fluoride-activated levels of adenylate cyclase were altered in membranes from cells cultured in the fields; however, the same membrane preparations exhibited markedly inhibited responses to parathyroid hormone. The fields blocked the inhibitory effects of the hormone on collagen synthesis by MMB-1 cells. However, there was no effect on the inhibition of collagen synthesis by 1,25-dihydroxyvitamin D3, which is believed to act primarily by a nuclear, rather than by a membrane-dependent, mechanism. No significant differences were noted between effects of the two fields, one generating continuous pulse trains (72 Hz) and the other generating recurrent bursts (15 Hz) of shorter pulses. We hypothesize that these field effects are mediated primarily at the plasma membrane of osteoblasts, either by interference with hormone-receptor interactions or by blocking of receptor-cyclase coupling in the membrane. These responses occurred with induced extracellular fields of 1 mV/cm or less, even though transmembrane potential gradients are typically 105 V/cm. PMID:6287472

  1. GABAB and adenosine receptors mediate enhancement of the K+ current, IAHP, by reducing adenylyl cyclase activity in rat CA3 hippocampal neurons.

    PubMed

    Gerber, U; Gähwiler, B H

    1994-11-01

    1. Gamma-aminobuturic acid-B (GABAB) and adenosine A1 receptors, which are expressed in hippocampal pyramidal cells, are linked to pertussis toxin-sensitive G-proteins known to be coupled negatively to the enzyme adenylyl cyclase. This study investigates the electrophysiological consequences of adenylyl cyclase inhibition in response to stimulation of these receptors. 2. Single-electrode voltage-clamp recordings were obtained from CA3 pyramidal cells in rat hippocampal slice cultures in presence of tetrodotoxin. The calcium-dependent potassium current (IAHP), which is very sensitive to intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP), was used as an electrophysiological indicator of adenylyl cyclase activity. 3. Application of baclofen (10 microM), a selective agonist at GABAB receptors, or adenosine (50 microM) each resulted in a transient decrease followed by a significant enhancement in the amplitude of evoked IAHP. The initial reduction in amplitude of IAHP probably reflects inadequacies in voltage clamp of electronically distant dendritic sites, due to the shunting caused by concomitant activation of potassium conductance by baclofen/adenosine. Comparable increases in membrane conductance in response to the GABAA agonist, muscimol, caused a similar reduction in IAHP. The enhancement of IAHP is consistent with an inhibition of constitutively active adenylyl cyclase. 4. The receptor mediating the responses to adenosine was identified as belonging to the A1 subtype on the basis of its sensitivity to the selective antagonist 8-cyclopentyl-1,3-dipropylxanthine.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Neutrophil beta-adrenergic receptor responses are potentiated by acute exposure to phorbol ester without changes in receptor distribution or coupling

    SciTech Connect

    Kilfeather, S.A.; Stein, M.; O'Malley, K. )

    1991-01-01

    Exposure to the phorbol ester, phorbol 12-myristate, 13-acetate 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{mu}M) 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-actylgylcerol (OAG) (50 {mu}M) also elevated beta-receptor responses, but 4beta-phorbol (100nM), lacking the capacity to activate PMA, was ineffective. Short-term exposure to the peptide n-formylmethionine leucyl-phenylalanine (FMLP, 1 {mu}M) 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 (H{sub 7}). PMA had no apparent effect on beta-receptor agonist-affinity, distribution between cell-surface and internalized compartments, or the capacity of ISO to induce beta-receptor internalization. Responses to FSK or ISO in terms of fold-stimulation of basal cyclic AMP accumulation int he presence of PMA were not elevated by PMA.

  3. The tcmVI region of the tetracenomycin C biosynthetic gene cluster of Streptomyces glaucescens encodes the tetracenomycin F1 monooxygenase, tetracenomycin F2 cyclase, and, most likely, a second cyclase.

    PubMed Central

    Summers, R G; Wendt-Pienkowski, E; Motamedi, H; Hutchinson, C R

    1993-01-01

    Certain mutations in the tcmVI region of the Streptomyces glaucescens chromosome affect formation of the D ring of the polyketide antibiotic tetracenomycin C (TCM C). This region lies immediately upstream from the TCM C polyketide synthase genes (tcmKLM), and the nucleotide sequence reveals the presence of three small genes, tcmH, tcmI, and tcmJ. On the basis of the phenotypes of mutants and the effects of these genes, when coupled on a plasmid with the tcmKLMN177 genes (tcmN177 is a 3'-truncated version of tcmN), on the production of TCM intermediates in a TCM- mutant, the tcmH gene encodes the C-5 monooxygenase that converts TCM F1 to TCM D3, the tcmI gene encodes the D-ring cyclase that converts TCM F2 to TCM F1 (mutations in this gene are responsible for the type VI phenotype), and the tcmJ gene most likely encodes the B-ring cyclase that acts in the biosynthesis of TCM F2. Furthermore, it appears that the N-terminal domain of the tcmN gene product (encoded by the tcmN177 gene) acts later in the biosynthesis of TCM F2 than the product of tcmJ, suggesting that the N-terminal domain of the TcmN protein is the C-ring cyclase. Images PMID:8244926

  4. Adenylyl cyclase 3 haploinsufficiency confers susceptibility to diet-induced obesity and insulin resistance in mice

    PubMed Central

    Tong, Tao; Shen, Ying; Lee, Han-Woong; Yu, Rina; Park, Taesun

    2016-01-01

    Adenylyl cyclase 3 (Adcy3), a member of the mammalian adenylyl cyclase family responsible for generating the second messenger cAMP, has long been known to play an essential role in olfactory signal transduction. Here, we demonstrated that Adcy3 heterozygous null mice displayed increased visceral adiposity in the absence of hyperphagia and developed abnormal metabolic features characterized by impaired insulin sensitivity, dyslipidemia, and increased plasma levels of proinflammatory cytokines on both chow and high-fat diet (HFD). Of note, HFD decreased the Adcy3 expression in white adipose tissue, liver, and muscle. We also report for the first time that Adcy3 haploinsufficiency resulted in reduced expression of genes involved in thermogenesis, fatty acid oxidation, and insulin signaling, with enhanced expression of genes related to adipogenesis in peripheral tissues of mice. In conclusion, these findings suggest that cAMP signals generated by Adcy3 in peripheral tissues may play a pivotal role in modulating obesity and insulin sensitivity. PMID:27678003

  5. Bicarbonate-sensing soluble adenylyl cyclase is an essential sensor for acid/base homeostasis

    PubMed Central

    Tresguerres, Martin; Parks, Scott K.; Salazar, Eric; Levin, Lonny R.; Goss, Greg G.; Buck, Jochen

    2009-01-01

    pH homeostasis is essential for life, yet it remains unclear how animals sense their systemic acid/base (A/B) status. Soluble adenylyl cyclase (sAC) is an evolutionary conserved signaling enzyme that produces the second messenger cAMP in response to bicarbonate ions (HCO3−). We cloned the sAC ortholog from the dogfish, a shark that regulates blood A/B by absorbing and secreting protons (H+) and HCO3− at its gills. Similar to mammalian sAC, dogfish soluble adenylyl cyclase (dfsAC) is activated by HCO3− and can be inhibited by two structurally and mechanistically distinct small molecule inhibitors. dfsAC is expressed in the gill epithelium, where the subset of base-secreting cells resides. Injection of inhibitors into animals under alkaline stress confirmed that dfsAC is essential for maintaining systemic pH and HCO3− levels in the whole organism. One of the downstream effects of dfsAC is to promote the insertion of vacuolar proton pumps into the basolateral membrane to absorb H+ into the blood. sAC orthologs are present throughout metazoans, and mammalian sAC is expressed in A/B regulatory organs, suggesting that systemic A/B sensing via sAC is widespread in the animal kingdom. PMID:20018667

  6. Bicarbonate-sensing soluble adenylyl cyclase is an essential sensor for acid/base homeostasis.

    PubMed

    Tresguerres, Martin; Parks, Scott K; Salazar, Eric; Levin, Lonny R; Goss, Greg G; Buck, Jochen

    2010-01-05

    pH homeostasis is essential for life, yet it remains unclear how animals sense their systemic acid/base (A/B) status. Soluble adenylyl cyclase (sAC) is an evolutionary conserved signaling enzyme that produces the second messenger cAMP in response to bicarbonate ions (HCO(3)(-)). We cloned the sAC ortholog from the dogfish, a shark that regulates blood A/B by absorbing and secreting protons (H(+)) and HCO(3)(-) at its gills. Similar to mammalian sAC, dogfish soluble adenylyl cyclase (dfsAC) is activated by HCO(3)(-) and can be inhibited by two structurally and mechanistically distinct small molecule inhibitors. dfsAC is expressed in the gill epithelium, where the subset of base-secreting cells resides. Injection of inhibitors into animals under alkaline stress confirmed that dfsAC is essential for maintaining systemic pH and HCO(3)(-) levels in the whole organism. One of the downstream effects of dfsAC is to promote the insertion of vacuolar proton pumps into the basolateral membrane to absorb H(+) into the blood. sAC orthologs are present throughout metazoans, and mammalian sAC is expressed in A/B regulatory organs, suggesting that systemic A/B sensing via sAC is widespread in the animal kingdom.

  7. Hypoxic Vasospasm Mediated by cIMP: When Soluble Guanylyl Cyclase Turns Bad

    PubMed Central

    Chen, Zhengju; Leung, Susan W. S.; Vanhoutte, Paul M.

    2015-01-01

    Abstract: 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 Ca2+ 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. PMID:25264755

  8. Modulation of a pre-existing conformational equilibrium tunes adenylate kinase activity.

    PubMed

    Ådén, Jörgen; Verma, Abhinav; Schug, Alexander; Wolf-Watz, Magnus

    2012-10-10

    Structural plasticity is often required for distinct microscopic steps during enzymatic reaction cycles. Adenylate kinase from Escherichia coli (AK(eco)) populates two major conformations in solution; the open (inactive) and closed (active) state, and the overall turnover rate is inversely proportional to the lifetime of the active conformation. Therefore, structural plasticity is intimately coupled to enzymatic turnover in AK(eco). Here, we probe the open to closed conformational equilibrium in the absence of bound substrate with NMR spectroscopy and molecular dynamics simulations. The conformational equilibrium in absence of substrate and, in turn, the turnover number can be modulated with mutational- and osmolyte-driven perturbations. Removal of one hydrogen bond between the ATP and AMP binding subdomains results in a population shift toward the open conformation and a resulting increase of k(cat). Addition of the osmolyte TMAO to AK(eco) results in population shift toward the closed conformation and a significant reduction of k(cat). The Michaelis constants (K(M)) scale with the change in k(cat), which follows from the influence of the population of the closed conformation for substrate binding affinity. Hence, k(cat) and K(M) are mutually dependent, and in the case of AK(eco), any perturbation that modulates k(cat) is mirrored with a proportional response in K(M). Thus, our results demonstrate that the equilibrium constant of a pre-existing conformational equilibrium directly affects enzymatic catalysis. From an evolutionary perspective, our findings suggest that, for AK(eco), there exists ample flexibility to obtain a specificity constant (k(cat)/K(M)) that commensurate with the exerted cellular selective pressure.

  9. Molecular cloning and characterization of a rat homolog of CAP, the adenylyl cyclase-associated protein from Saccharomyces cerevisiae.

    PubMed

    Zelicof, A; Gatica, J; Gerst, J E

    1993-06-25

    We have isolated a rat cDNA whose expression suppresses the physiological consequences of the chromosomal disruption of CAP, the gene encoding the adenylyl cyclase-associated protein of Saccharomyces cerevisiae. Yeast CAP is a bifunctional protein: the NH2 terminus is necessary and sufficient for cellular responsiveness to activated RAS proteins, while the COOH terminus is required for normal cellular morphology and growth control. The rat MCH1 cDNA encodes a protein of 474 amino acids that is 36% identical to S. cerevisiae CAP and is capable of suppressing the loss of the COOH-terminal functions of CAP when expressed in yeast. The MCH1 protein therefore appears to be a structural and functional homolog of the yeast cyclase-associated proteins. Northern analysis of MCH1 gene expression shows it to be constitutively expressed in all cell and tissue types examined. The cloning of a rat homolog of CAP, in addition to the cloning of a human CAP homolog by Matviw et al. (Matviw, H., Yu, G., and Young, D. (1992) Mol. Cell. Biol. 12, 5033-5040), demonstrates that both cyclase-associated proteins and their functions may have evolved with mammalian cells.

  10. Adenylyl cyclase G, an osmosensor controlling germination of Dictyostelium spores.

    PubMed

    van Es, S; Virdy, K J; Pitt, G S; Meima, M; Sands, T W; Devreotes, P N; Cotter, D A; Schaap, P

    1996-09-27

    Dictyostelium cells express a G-protein-coupled adenylyl cyclase, ACA, during aggregation and an atypical adenylyl cyclase, ACG, in mature spores. The ACG gene was disrupted by homologous recombination. acg- cells developed into normal fruiting bodies with viable spores, but spore germination was no longer inhibited by high osmolarity, a fairly universal constraint for spore and seed germination. ACG activity, measured in aca-/ACG cells, was strongly stimulated by high osmolarity with optimal stimulation occurring at 200 milliosmolar. RdeC mutants, which display unrestrained protein kinase A (PKA) activity and a cell line, which overexpresses PKA under a prespore specific promoter, germinate very poorly, both at high and low osmolarity. These data indicate that ACG is an osmosensor controlling spore germination through activation of protein kinase A.

  11. Intracellular cAMP signaling by soluble adenylyl cyclase.

    PubMed

    Tresguerres, Martin; Levin, Lonny R; Buck, Jochen

    2011-06-01

    Soluble adenylyl cyclase (sAC) is a recently identified source of the ubiquitous second messenger cyclic adenosine 3',5' monophosphate (cAMP). sAC is distinct from the more widely studied source of cAMP, the transmembrane adenylyl cyclases (tmACs); its activity is uniquely regulated by bicarbonate anions, and it is distributed throughout the cytoplasm and in cellular organelles. Due to its unique localization and regulation, sAC has various functions in a variety of physiological systems that are distinct from tmACs. In this review, we detail the known functions of sAC, and we reassess commonly held views of cAMP signaling inside cells.

  12. Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

    SciTech Connect

    Zhang, Z.; Swaminathan, S.; Zhou, R.; Sauder, J. M.; Tonge, P. J.; Burley, S. K.

    2011-02-18

    Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

  13. Structural and Functional Studies of Fatty Acyl Adenylate Ligases from E. coli and L. pneumophila

    SciTech Connect

    Z Zhang; R Zhou; J Sauder; P Tonge; S Burley; S Swaminathan

    2011-12-31

    Fatty acyl-AMP ligase (FAAL) is a new member of a family of adenylate-forming enzymes that were recently discovered in Mycobacterium tuberculosis. They are similar in sequence to fatty acyl-coenzyme A (CoA) ligases (FACLs). However, while FACLs perform a two-step catalytic reaction, AMP ligation followed by CoA ligation using ATP and CoA as cofactors, FAALs produce only the acyl adenylate and are unable to perform the second step. We report X-ray crystal structures of full-length FAAL from Escherichia coli (EcFAAL) and FAAL from Legionella pneumophila (LpFAAL) bound to acyl adenylate, determined at resolution limits of 3.0 and 1.85 {angstrom}, respectively. The structures share a larger N-terminal domain and a smaller C-terminal domain, which together resemble the previously determined structures of FAAL and FACL proteins. Our two structures occur in quite different conformations. EcFAAL adopts the adenylate-forming conformation typical of FACLs, whereas LpFAAL exhibits a unique intermediate conformation. Both EcFAAL and LpFAAL have insertion motifs that distinguish them from the FACLs. Structures of EcFAAL and LpFAAL reveal detailed interactions between this insertion motif and the interdomain hinge region and with the C-terminal domain. We suggest that the insertion motifs support sufficient interdomain motions to allow substrate binding and product release during acyl adenylate formation, but they preclude CoA binding, thereby preventing CoA ligation.

  14. Cure and curse: E. coli heat-stable enterotoxin and its receptor guanylyl cyclase C.

    PubMed

    Weiglmeier, Philipp R; Rösch, Paul; Berkner, Hanna

    2010-09-01

    Enterotoxigenic Escherichia coli (ETEC) associated diarrhea is responsible for roughly half a million deaths per year, the majority taking place in developing countries. The main agent responsible for these diseases is the bacterial heat-stable enterotoxin STa. STa is secreted by ETEC and after secretion binds to the intestinal receptor guanylyl cyclase C (GC-C), thus triggering a signaling cascade that eventually leads to the release of electrolytes and water in the intestine. Additionally, GC-C is a specific marker for colorectal carcinoma and STa is suggested to have an inhibitory effect on intestinal carcinogenesis. To understand the conformational events involved in ligand binding to GC-C and to devise therapeutic strategies to treat both diarrheal diseases and colorectal cancer, it is paramount to obtain structural information on the receptor ligand system. Here we summarize the currently available structural data and report on physiological consequences of STa binding to GC-C in intestinal epithelia and colorectal carcinoma cells.

  15. Cyclic adenosine monophosphate-dependent vascular responses to purinergic agonists adenosine triphosphate and uridine triphosphate in the anesthetized mouse.

    PubMed

    Shah, Mrugeshkumar K; Kadowitz, Philip J

    2002-01-01

    The mechanism by which purinergic agonist adenosine triphosphate (ATP) and uridine triphosphate (UTP) decrease systemic arterial pressure in the anesthetized mouse was investigated. Intravenous injections of adenosine triphosphate (ATP) and uridine triphosphate (UTP) produced dose-dependent decreases in systemic blood pressure in the mouse. The order of potency was ATP > UTP. Vasodilator responses to ATP and UTP were altered by the cyclic adenosine monophosphate (cAMP) phosphodiesterase inhibitor rolipram. The vascular responses to ATP and UTP were not altered by a nitric oxide synthase inhibitor, a cyclooxygenase inhibitor, a cGMP phosphodiesterase inhibitor, or a particular P2 receptor antagonist. These data suggest that ATP and UTP cause a decrease in systemic arterial pressure in the mouse via a cAMP-dependent pathway via a novel P2 receptor linked to adenylate cyclase and that nitric oxide release, prostaglandin synthesis, cGMP, and P2X1, P2Y1, and P2Y4 receptors play little or no role in the vascular effects of these purinergic agonists in the mouse.

  16. Potent Anti-Trypanosoma cruzi Activities of Oxidosqualene Cyclase Inhibitors

    PubMed Central

    Buckner, Frederick S.; Griffin, John H.; Wilson, Aaron J.; Van Voorhis, Wesley C.

    2001-01-01

    Trypanosoma cruzi is the protozoan agent that causes Chagas' disease, a major health problem in Latin America. Better drugs are needed to treat infected individuals. The sterol biosynthesis pathway is a potentially excellent target for drug therapy against T. cruzi. In this study, we investigated the antitrypanosomal activities of a series of compounds designed to inhibit a key enzyme in sterol biosynthesis, oxidosqualene cyclase. This enzyme converts 2,3-oxidosqualene to the tetracyclic product, lanosterol. The lead compound, N-(4E,8E)-5,9, 13-trimethyl-4,8, 12-tetradecatrien-1-ylpyridinium, is an electron-poor aromatic mimic of a monocyclized transition state or high-energy intermediate formed from oxidosqualene. This compound and 27 related compounds were tested against mammalian-stage T. cruzi, and 12 inhibited growth by 50% at concentrations below 25 nM. The lead compound was shown to cause an accumulation of oxidosqualene and decreased production of lanosterol and ergosterol, consistent with specific inhibition of the oxidosqualene cyclase. The data demonstrate potent anti-T. cruzi activity associated with inhibition of oxidosqualene cyclase. PMID:11257036

  17. 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.

  18. Role of neutrophils in response to Bordetella pertussis infection in mice.

    PubMed

    Andreasen, Charlotte; Carbonetti, Nicholas H

    2009-03-01

    Pertussis is an acute respiratory disease caused by the bacterium Bordetella pertussis, for which humans are the only known reservoir. During infection, B. pertussis releases several toxins, including pertussis toxin (PT) and adenylate cyclase toxin (ACT), which have both been shown to play roles in promoting bacterial growth during early infection in a mouse model. Furthermore, in vitro and in vivo studies suggest that PT and ACT affect neutrophil chemotaxis and/or function, thereby altering the innate immune response. In this study we depleted animals of neutrophils to investigate whether neutrophils play a protective role during B. pertussis infection in mice. In addition, by infection with toxin-deficient strains, we investigated whether neutrophils are the main targets for PT and/or ACT activity in promoting bacterial growth. Surprisingly, we found no role for neutrophils during B. pertussis infection in naïve mice. However, in previously infected (immune) mice or in mice receiving immune serum, we observed a significant role for neutrophils during infection. Furthermore, in this immune mouse model our evidence indicates that neutrophils appear to be the main target cells for ACT, but not for PT.

  19. Opioid agonists binding and responses in SH-SY5Y cells

    NASA Technical Reports Server (NTRS)

    Costa, E. M.; Hoffmann, B. B.; Loew, G. H.

    1992-01-01

    SH-SY5Y (human neuroblastoma) cultured cells, known to have mu-opioid receptors, have been used to assess and compare the ability of eight representative mu-selective compounds from diverse opioid families to recognize and activate these receptors. A wide range of receptor affinities spanning a factor of 10,000 was found between the highest affinity fentanyl analogs (Ki = 0.1nM) and the lowest affinity analog, meperidine (Ki = 1 microM). A similar range was found for inhibition of PGE1-stimulated cAMP accumulation with a rank order of activities that closely paralleled binding affinities. Maximum inhibition of cAMP accumulation by each compound was about 80%. Maximum stimulation of GTPase activity (approximately 50%) was also similar for all compounds except the lowest affinity meperidine. Both effects were naloxone reversible. These results provide further evidence that mu-receptors are coupled to inhibition of adenylate cyclase and that the SH-SY5Y cell line is a good system for assessment of mu-agonists functional responses.

  20. 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.

  1. Cell surface adrenergic receptor stimulation modifies the endothelial response to SIRS. Systemic Inflammatory Response Syndrome.

    PubMed

    Tighe, D; Moss, R; Bennett, D

    1996-11-01

    The complex pathway seen in patients with the systemic inflammatory response syndrome (SIRS) does not readily respond to mediator blockade. All such trials conducted in SIRS patients have shown no benefit in reducing mortality. We have shown experimentally that in sepsis, the administration of beta 2-adrenoceptor agonists reduces hepatic cellular injury, whereas administration of an alpha 1-adrenoceptor agonist increases hepatic cellular injury. Inflammatory mediators can cause a dose-related reversible change in target endothelial cells (ECs). There is a substantial body of literature describing the anti-inflammatory effects of beta 2-adrenoceptor agonists. They reduce both the increased permeability and the production of inflammatory mediators from ECs. Cellular transduction processes are involved when adrenergic receptor agonists modify either the anti-inflammatory or proinflammatory response to sepsis in ECs. Inflammatory mediators and alpha 1-adrenoceptor agonists stimulate their trimeric G protein-linked receptors to produce diacylglycerol (DAG) and increase the intracellular concentration of calcium. DAG is involved in the production of both inflammatory proteins and lipids. In addition, mitogen-activated protein kinase (MAPK) is activated which is also involved in the production of inflammatory proteins and lipids. beta 2-adrenoceptor agonists activate their trimeric G protein-linked receptors to produce the stimulatory G protein (Gs). Gs stimulates adenyl cyclase to form cyclic adenosine monophosphate (cAMP) and activate protein kinase A (PKA). PKA is involved in activating gene transcription agents to produce anti-inflammatory proteins such as interleukin-10. PKA also inhibits phospholipase C and MAPK. Although promising, the use of beta-adrenoceptor agonists or agonists that increase cellular cAMP to activate the cells' endogenous anti-inflammatory pathway requires further study.

  2. Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.

    PubMed

    Dong, Qian; Ernst, Sarah E; Ostedgaard, Lynda S; Shah, Viral S; Ver Heul, Amanda R; Welsh, Michael J; Randak, Christoph O

    2015-05-29

    The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia.

  3. Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia*

    PubMed Central

    Dong, Qian; Ernst, Sarah E.; Ostedgaard, Lynda S.; Shah, Viral S.; Ver Heul, Amanda R.; Welsh, Michael J.; Randak, Christoph O.

    2015-01-01

    The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P1,P5-di(adenosine-5′) pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5′-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5′-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl− channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. PMID:25887396

  4. Key Role of the Adenylate Moiety and Integrity of the Adenylate-Binding Site for the NAD(+)/H Binding to Mitochondrial Apoptosis-Inducing Factor.

    PubMed

    Sorrentino, Luca; Calogero, Alessandra Maria; Pandini, Vittorio; Vanoni, Maria Antonietta; Sevrioukova, Irina F; Aliverti, Alessandro

    2015-12-01

    Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with pro-life and pro-death activities, which plays critical roles in mitochondrial energy metabolism and caspase-independent apoptosis. Defects in AIF structure or expression can cause mitochondrial abnormalities leading to mitochondrial defects and neurodegeneration. The mechanism of AIF-induced apoptosis was extensively investigated, whereas the mitochondrial function of AIF is poorly understood. A unique feature of AIF is the ability to form a tight, air-stable charge-transfer (CT) complex upon reaction with NADH and to undergo a conformational switch leading to dimerization, proposed to be important for its vital and lethal functions. Although some aspects of interaction of AIF with NAD(+)/H have been analyzed, its precise mechanism is not fully understood. We investigated how the oxidized and photoreduced wild-type and G307A and -E variants of murine AIF associate with NAD(+)/H and nicotinamide mononucleotide (NMN(+)/H) to determine the role of the adenylate moiety in the binding process. Our results indicate that (i) the adenylate moiety of NAD(+)/H is crucial for the association with AIF and for the subsequent structural reorganization of the complex, but not for protein dimerization, (ii) FAD reduction rather than binding of NAD(+)/H to AIF initiates conformational rearrangement, and (iii) alteration of the adenylate-binding site by the G307E (equivalent to a pathological G308E mutation in human AIF) or G307A replacements decrease the affinity and association rate of NAD(+)/H, which, in turn, perturbs CT complex formation and protein dimerization but has no influence on the conformational switch in the regulatory peptide.

  5. The Crystal Structure of the Adenylation Enzyme VinN Reveals a Unique β-Amino Acid Recognition Mechanism*

    PubMed Central

    Miyanaga, Akimasa; Cieślak, Jolanta; Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2014-01-01

    Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp230 residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes. PMID:25246523

  6. The crystal structure of the adenylation enzyme VinN reveals a unique β-amino acid recognition mechanism.

    PubMed

    Miyanaga, Akimasa; Cieślak, Jolanta; Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2014-11-07

    Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp(230) residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes.

  7. Role for the membrane receptor guanylyl cyclase-C in attention deficiency and hyperactive behavior.

    PubMed

    Gong, Rong; Ding, Cheng; Hu, Ji; Lu, Yao; Liu, Fei; Mann, Elizabeth; Xu, Fuqiang; Cohen, Mitchell B; Luo, Minmin

    2011-09-16

    Midbrain dopamine neurons regulate many important behavioral processes, and their dysfunctions are associated with several human neuropsychiatric disorders such as attention deficit hyperactivity disorder (ADHD) and schizophrenia. Here, we report that these neurons in mice selectively express guanylyl cyclase-C (GC-C), a membrane receptor previously thought to be expressed mainly in the intestine. GC-C activation potentiates the excitatory responses mediated by glutamate and acetylcholine receptors via the activity of guanosine 3',5'-monophosphate-dependent protein kinase (PKG). Mice in which GC-C has been knocked out exhibit hyperactivity and attention deficits. Moreover, their behavioral phenotypes are reversed by ADHD therapeutics and a PKG activator. These results indicate important behavioral and physiological functions for the GC-C/PKG signaling pathway within the brain and suggest new therapeutic targets for neuropsychiatric disorders related to the malfunctions of midbrain dopamine neurons.

  8. 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.

  9. Cellular basis for the olfactory response to nicotine.

    PubMed

    Bryant, Bruce; Xu, Jiang; Audige, Valery; Lischka, Fritz W; Rawson, Nancy E

    2010-03-17

    Smokers regulate their smoking behavior on the basis of sensory stimuli independently of the pharmacological effects of nicotine (Rose J. E., et al. (1993) Pharmacol., Biochem. Behav.44 (4), 891-900). A better understanding of sensory mechanisms underlying smoking behavior may help to develop more effective smoking alternatives. Olfactory stimulation by nicotine makes up a considerable part of the flavor of tobacco smoke, yet our understanding of the cellular mechanisms responsible for olfactory detection of nicotine remains incomplete. We used biophysical methods to characterize the nicotine sensitivity and response mechanisms of neurons from olfactory epithelium. In view of substantial differences in the olfactory receptor repertoire between rodent and human (Mombaerts P. (1999) Annu. Rev. Neurosci.22, 487-509), we studied biopsied human olfactory sensory neurons (OSNs), cultured human olfactory cells (Gomez G., et al. (2000) J. Neurosci. Res.62 (5), 737-749), and rat olfactory neurons. Rat and human OSNs responded to S(-)-nicotine with a concentration dependent influx of calcium and activation of adenylate cyclase. Some rat OSNs displayed some stereoselectivity, with neurons responding to either enantiomer alone or to both. Freshly biopsied and primary cultured human olfactory neurons were less stereoselective. Nicotinic cholinergic antagonists had no effect on the responses of rat or human OSNs to nicotine. Patch clamp recording of rat OSNs revealed a nicotine-activated, calcium-sensitive nonspecific cation channel. These results indicate that nicotine activates a canonical olfactory receptor pathway rather than nicotinic cholinergic receptors on OSNs. Further, because the nicotine-sensitive mechanisms of rodents appear generally similar to those of humans, this animal model is an appropriate one for studies of nicotine sensation.

  10. Multiforms of mammalian adenylate kinase and its monoclonal antibody against AK1.

    PubMed

    Kurokawa, Y; Takenaka, H; Sumida, M; Oka, K; Hamada, M; Kuby, S A

    1990-01-01

    An attempt has been made to determine the intracellular distribution of the multiforms of the adenylate kinase (AK) isoenzymes in mammalian tissues, to shed some light on their physiological roles, especially in energy metabolism. The adenylate kinase zymograms obtained from isoelectric focusing yielded two typical isoform patterns: (1) with a pI greater than or equal to 9 and 8.6, specific for bovine skeletal muscle, heart, aorta and brain, and (2) with a pI = 7.9 and 7.1, specific for liver and kidney. Pattern (1) was attributed to the cytosolic isoenzyme (AK1) as demonstrated by immunostaining with anti-AK1. Pattern (2) was attributed to the mitochondrial isoenzyme (AK2). These results were largely confirmed by chromatofocusing experiments. The AK1 isoenzyme was partially purified from the cytosol fraction of bovine aortic smooth muscle and had an apparent Mr of 23.5 kilodaltons. Its kinetic features are discussed from a comparative standpoint. Finally, the human serum AK1 isoform was also detected by Western blotting with a monoclonal antibody directed against crystalline porcine muscle AK1. These results are to form the basis of further studies on the 'aberrant' adenylate kinase isoenzyme from the serum of Duchenne muscular dystrophics.

  11. Molecular and functional characterization of a Trypanosoma cruzi nuclear adenylate kinase isoform.

    PubMed

    Cámara, María de los Milagros; Bouvier, León A; Canepa, Gaspar E; Miranda, Mariana R; Pereira, Claudio A

    2013-01-01

    Trypanosoma cruzi, the etiological agent of Chagas' disease, is an early divergent eukaryote in which control of gene expression relies mainly in post-transcriptional mechanisms. Transcription levels are globally up and down regulated during the transition between proliferating and non-proliferating life-cycle stages. In this work we characterized a nuclear adenylate kinase isoform (TcADKn) that is involved in ribosome biogenesis. Nuclear adenylate kinases have been recently described in a few organisms, being all related to RNA metabolism. Depending on active transcription and translation, TcADKn localizes in the nucleolus or the cytoplasm. A non-canonical nuclear localization signal was mapped towards the N-terminal of the protein, being the phosphate-binding loop essential for its localization. In addition, TcADKn nuclear exportation depends on the nuclear exportation adapter CRM1. TcADKn nuclear shuttling is governed by nutrient availability, oxidative stress and by the equivalent in T. cruzi of the mammalian TOR (Target of Rapamycin) pathway. One of the biological functions of TcADKn is ribosomal 18S RNA processing by direct interaction with ribosomal protein TcRps14. Finally, TcADKn expression is regulated by its 3' UTR mRNA. Depending on extracellular conditions, cells modulate protein translation rates regulating ribosome biogenesis and nuclear adenylate kinases are probably key components in these processes.

  12. Molecular and Functional Characterization of a Trypanosoma cruzi Nuclear Adenylate Kinase Isoform

    PubMed Central

    Cámara, María de los Milagros; Bouvier, León A.; Canepa, Gaspar E.; Miranda, Mariana R.; Pereira, Claudio A.

    2013-01-01

    Trypanosoma cruzi, the etiological agent of Chagas' disease, is an early divergent eukaryote in which control of gene expression relies mainly in post-transcriptional mechanisms. Transcription levels are globally up and down regulated during the transition between proliferating and non-proliferating life-cycle stages. In this work we characterized a nuclear adenylate kinase isoform (TcADKn) that is involved in ribosome biogenesis. Nuclear adenylate kinases have been recently described in a few organisms, being all related to RNA metabolism. Depending on active transcription and translation, TcADKn localizes in the nucleolus or the cytoplasm. A non-canonical nuclear localization signal was mapped towards the N-terminal of the protein, being the phosphate-binding loop essential for its localization. In addition, TcADKn nuclear exportation depends on the nuclear exportation adapter CRM1. TcADKn nuclear shuttling is governed by nutrient availability, oxidative stress and by the equivalent in T. cruzi of the mammalian TOR (Target of Rapamycin) pathway. One of the biological functions of TcADKn is ribosomal 18S RNA processing by direct interaction with ribosomal protein TcRps14. Finally, TcADKn expression is regulated by its 3′ UTR mRNA. Depending on extracellular conditions, cells modulate protein translation rates regulating ribosome biogenesis and nuclear adenylate kinases are probably key components in these processes. PMID:23409202

  13. A non-canonical peptide synthetase adenylates 3-methyl-2-oxovaleric acid for auriculamide biosynthesis

    PubMed Central

    Braga, Daniel; Hoffmeister, Dirk

    2016-01-01

    Auriculamide is the first natural product known from the predatory bacterium Herpetosiphon aurantiacus. It is composed of three unusual building blocks, including the non-proteinogenic amino acid 3-chloro-L-tyrosine, the α-hydroxy acid L-isoleucic acid, and a methylmalonyl-CoA-derived ethane unit. A candidate genetic locus for auriculamide biosynthesis was identified and encodes four enzymes. Among them, the non-canonical 199 kDa four-domain nonribosomal peptide synthetase, AulA, is extraordinary in that it features two consecutive adenylation domains. Here, we describe the functional characterization of the recombinantly produced AulA. The observed activation of 3-methyl-2-oxovaleric acid by the enzyme supports the hypothesis that it participates in the biosynthesis of auriculamide. An artificially truncated version of AulA that lacks the first adenylation domain activated this substrate like the full-length enzyme which shows that the first adenylation domain is dispensable. Additionally, we provide evidence that the enzyme tolerates structural variation of the substrate. α-Carbon substituents significantly affected the substrate turnover. While all tested aliphatic α-keto acids were accepted by the enzyme and minor differences in chain size and branches did not interfere with the enzymatic activity, molecules with methylene α-carbons led to low turnover. Such enzymatic plasticity is an important attribute to help in the perpetual search for novel molecules and to access a greater structural diversity by mutasynthesis. PMID:28144348

  14. The intact CFTR protein mediates ATPase rather than adenylate kinase activity.

    PubMed

    Ramjeesingh, Mohabir; Ugwu, Francisca; Stratford, Fiona L L; Huan, Ling-Jun; Li, Canhui; Bear, Christine E

    2008-06-01

    The two NBDs (nucleotide-binding domains) of ABC (ATP-binding-cassette) proteins function in a complex to mediate ATPase activity and this activity has been linked to their regulated transport activity. A similar model has been proposed for CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel defective in cystic fibrosis, wherein ATP binding and hydrolysis regulate the channel gate. Recently, it was shown that the individual NBDs isolated from CFTR primarily mediate adenylate kinase activity, raising the possibility that this activity may also contribute to gating of the CFTR channel. However, this present study shows that whereas the isolated NBDs exhibit adenylate kinase activity, the full-length purified and reconstituted CFTR protein functions as an ATPase, arguing that the enzymatic activity of the NBDs is dependent on their molecular context and appropriate domain-domain assembly. As expected, the disease-causing mutant bearing a mutation in the ABC signature motif, CFTR-G551D, exhibited a markedly reduced ATPase activity. Furthermore, mutation of the putative catalytic base in CFTR caused a reduction in ATPase activity, with the CFTR-E1371Q mutant supporting a low level of residual activity. Neither of these mutants exhibited detectable adenylate kinase activity. Together, these findings support the concept that the molecular mechanism of action of CFTR is dependent on ATP binding and hydrolysis, and that the structure of prokaryotic ABC ATPases provide a useful template for understanding their mechanism of action.

  15. Diguanylate cyclase NicD-based signalling mechanism of nutrient-induced dispersion by Pseudomonas aeruginosa.

    PubMed

    Basu Roy, Ankita; Sauer, Karin

    2014-11-01

    Dispersion enables the transition from the biofilm to the planktonic growth state in response to various cues. While several Pseudomonas aeruginosa proteins, including BdlA and the c-di-GMP phosphodiesterases DipA, RbdA, and NbdA, have been shown to be required for dispersion to occur, little is known about dispersion cue sensing and the signalling translating these cues into the modulation c-di-GMP levels to enable dispersion. Using glutamate-induced dispersion as a model, we report that dispersion-inducing nutrient cues are sensed via an outside-in signalling mechanism by the diguanylate cyclase NicD belonging to a family of seven transmembrane (7TM) receptors. NicD directly interacts with BdlA and the phosphodiesterase DipA, with NicD, BdlA, and DipA being part of the same pathway required for dispersion. Glutamate sensing by NicD results in NicD dephosphorylation and increased cyclase activity. Active NicD contributes to the non-processive proteolysis and activation of BdlA via phosphorylation and temporarily elevated c-di-GMP levels. BdlA, in turn, activates DipA, resulting in the overall reduction of c-di-GMP levels. Our results provide a basis for understanding the signalling mechanism based on NicD to induce biofilm dispersion that may be applicable to various biofilm-forming species and may have implications for the control of biofilm-related infections.

  16. Characterization of a novel sesquiterpene cyclase involved in (+)-caryolan-1-ol biosynthesis in Streptomyces griseus.

    PubMed

    Nakano, Chiaki; Horinouchi, Sueharu; Ohnishi, Yasuo

    2011-08-12

    Most terpenoids have been isolated from plants and fungi and only a few from bacteria. However, an increasing number of genome sequences indicate that bacteria possess a variety of terpenoid cyclase genes. We characterized a sesquiterpene cyclase gene (SGR2079, named gcoA) found in Streptomyces griseus. When expressed in Streptomyces lividans, gcoA directed production of a sesquiterpene, isolated and determined to be (+)-caryolan-1-ol using spectroscopic analyses. (+)-Caryolan-1-ol was also detected in the crude cell lysate of wild-type S. griseus but not in a gcoA knockout mutant, indicating that GcoA is a genuine (+)-caryolan-1-ol synthase. Enzymatic properties were characterized using N-terminally histidine-tagged GcoA, produced in Escherichia coli. As expected, incubation of the recombinant GcoA protein with farnesyl diphosphate yielded (+)-caryolan-1-ol. However, a small amount of another sesquiterpene was also detected. This was identified as the bicyclic sesquiterpene hydrocarbon (+)-β-caryophyllene by comparison with an authentic sample using GC-MS. Incorporation of a deuterium atom into the C-9 methylene of (+)-caryolan-1-ol in an in vitro GcoA reaction in deuterium oxide indicated that (+)-caryolan-1-ol was synthesized by a proton attack on the C-8/C-9 double bond of (+)-β-caryophyllene. Several β-caryophyllene synthases have been identified from plants, but these cannot synthesize caryolan-1-ol. Although caryolan-1-ol has been isolated previously from several plants, the enzyme responsible for its biosynthesis has not been identified previously. GcoA is thus the first known caryolan-1-ol synthase. Isolation of caryolan-1-ol from microorganisms is unprecedented.

  17. Stimulation of renin secretion by catecholamines is dependent on adenylyl cyclases 5 and 6.

    PubMed

    Aldehni, Fadi; Tang, Tong; Madsen, Kirsten; Plattner, Michael; Schreiber, Andrea; Friis, Ulla G; Hammond, H Kirk; Han, Pyung Lim; Schweda, Frank

    2011-03-01

    The sympathetic nervous system stimulates renin release from juxtaglomerular cells via the β-adrenoreceptor-cAMP pathway. Recent in vitro studies have suggested that the calcium-inhibited adenylyl cyclases (ACs) 5 and 6 possess key roles in the control of renin exocytosis. To investigate the relative contribution of AC5 and AC6 to the regulation of renin release in vivo we performed experiments using AC5 and AC6 knockout mice. Male AC5(-/-) mice exhibited normal plasma renin concentrations, renal renin synthesis (mRNA and renin content), urinary volume, and systolic blood pressure. In male AC6(-/-) mice, plasma renin concentration (AC6(-/-): 732 ± 119; AC6 (+/+): 436 ± 78 ng of angiotensin I per hour*mL(-1); P<0.05), and renin synthesis were stimulated associated with an increased excretion of dilute urine (1.55-fold; P<0.05) and reduced blood pressure (-10.6 mm Hg; P<0.001). Stimulation of plasma renin concentration by a single injection of the β-adrenoreceptor agonist isoproterenol (10 mg/kg IP) was significantly attenuated in AC5(-/-) (male: -20%; female: -33%) compared with wild-type mice in vivo. The mitigation of the plasma renin concentration response to isoproterenol was even more pronounced in AC6(-/-) (male: -63%; female: -50% versus AC6(+/+)). Similarly, the effects of isoproterenol, prostaglandin E2, and pituitary adenylyl cyclase-activating polypeptide on renin release from isolated perfused kidneys were attenuated to a higher extent in AC6(-/-) (-51% to -98% versus AC6(+/+)) than in AC5(-/-) (-31% to 46% versus AC5(+/+)). In conclusion, both AC5 and AC6 are involved in the stimulation of renin secretion in vivo, and AC6 is the dominant isoforms in this process.

  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. Nitric Oxide-Induced Conformational Changes in Soluble Guanylate Cyclase

    PubMed Central

    Underbakke, Eric S.; Iavarone, Anthony T.; Chalmers, Michael J.; Pascal, Bruce D.; Novick, Scott; Griffin, Patrick R.; Marletta, Michael A.

    2014-01-01

    SUMMARY Soluble guanylate cyclase (sGC) is the primary mediator of nitric oxide (NO) signaling. NO binds the sGC heme cofactor stimulating synthesis of the second messenger cyclic-GMP (cGMP). As the central hub of NO/cGMP signaling pathways, sGC is important in diverse physiological processes such as vasodilation and neurotransmission. Nevertheless, the mechanisms underlying NO-induced cyclase activation in sGC remain unclear. Here, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was employed to probe the NO-induced conformational changes of sGC. HDX-MS revealed NO-induced effects in several discrete regions. NO binding to the heme-NO/O2-binding (H-NOX) domain perturbs a signaling surface implicated in Per/Arnt/Sim (PAS) domain interactions. Furthermore, NO elicits striking conformational changes in the junction between the PAS and helical domains that propagate as perturbations throughout the adjoining helices. Ultimately, NO-binding stimulates the catalytic domain by contracting the active site pocket. Together, these conformational changes delineate an allosteric pathway linking NO-binding to activation of the catalytic domain. PMID:24560804

  20. Increased behavioral and neuronal responses to a hallucinogenic drug in PACAP heterozygous mutant mice.

    PubMed

    Hazama, Keisuke; Hayata-Takano, Atsuko; Uetsuki, Kazuki; Kasai, Atsushi; Encho, Naoki; Shintani, Norihito; Nagayasu, Kazuki; Hashimoto, Ryota; Reglodi, Dora; Miyakawa, Tsuyoshi; Nakazawa, Takanobu; Baba, Akemichi; Hashimoto, Hitoshi

    2014-01-01

    Accumulating evidence from human genetic studies implicates the pituitary adenylate cyclase-activating polypeptide (PACAP) gene as a risk factor for psychiatric disorders, including schizophrenia and stress-related diseases. Mice with homozygous disruption of the PACAP gene display profound behavioral and neurological abnormalities that are ameliorated with the atypical antipsychotic and dopamine D2 and serotonin (5-HT)2 antagonist risperidone and the 5-HT2 receptor antagonist ritanserin; however, the underlying mechanisms remain unknown. Here, we investigated if PACAP heterozygous mutant (PACAP(+/-)) mice, which appear behaviorally normal, are vulnerable to aversive stimuli. PACAP(+/-) mice were administered a 5-HT2 receptor agonist, (±)-2,5-dimethoxy-4-iodoamphetamine (DOI), a hallucinogenic drug, and their responses were compared with the littermate wild-type mice. After DOI injection, PACAP(+/-) mice showed increased head-twitch responses, while their behavior was normal after saline. DOI induced deficits in sensorimotor gating, as determined by prepulse inhibition, specifically in PACAP(+/-) mice. However, other 5-HT2 receptor-dependent responses, such as corticosterone release and hypothermia, were similarly observed in PACAP(+/-) and wild-type mice. c-Fos expression analysis, performed in various brain regions, revealed that the DOI-induced increase in the number of c-Fos-positive cells was more pronounced in 5-HT2A receptor-negative cells in the somatosensory cortex in PACAP(+/-) mice compared with wild-type mice. These results indicate that PACAP(+/-) mice exhibit specific vulnerability to DOI-induced deficits in cortical sensory function, such as exaggerated head-twitch responses and sensorimotor gating deficits. Our findings provide insight into the neural mechanisms underlying impaired behavioral responses in which 5-HT2 receptors are implicated.

  1. Increased Behavioral and Neuronal Responses to a Hallucinogenic Drug in PACAP Heterozygous Mutant Mice

    PubMed Central

    Kasai, Atsushi; Encho, Naoki; Shintani, Norihito; Nagayasu, Kazuki; Hashimoto, Ryota; Reglodi, Dora; Miyakawa, Tsuyoshi; Nakazawa, Takanobu; Baba, Akemichi; Hashimoto, Hitoshi

    2014-01-01

    Accumulating evidence from human genetic studies implicates the pituitary adenylate cyclase-activating polypeptide (PACAP) gene as a risk factor for psychiatric disorders, including schizophrenia and stress-related diseases. Mice with homozygous disruption of the PACAP gene display profound behavioral and neurological abnormalities that are ameliorated with the atypical antipsychotic and dopamine D2 and serotonin (5-HT)2 antagonist risperidone and the 5-HT2 receptor antagonist ritanserin; however, the underlying mechanisms remain unknown. Here, we investigated if PACAP heterozygous mutant (PACAP+/−) mice, which appear behaviorally normal, are vulnerable to aversive stimuli. PACAP+/− mice were administered a 5-HT2 receptor agonist, (±)-2,5-dimethoxy-4-iodoamphetamine (DOI), a hallucinogenic drug, and their responses were compared with the littermate wild-type mice. After DOI injection, PACAP+/− mice showed increased head-twitch responses, while their behavior was normal after saline. DOI induced deficits in sensorimotor gating, as determined by prepulse inhibition, specifically in PACAP+/− mice. However, other 5-HT2 receptor-dependent responses, such as corticosterone release and hypothermia, were similarly observed in PACAP+/− and wild-type mice. c-Fos expression analysis, performed in various brain regions, revealed that the DOI-induced increase in the number of c-Fos-positive cells was more pronounced in 5-HT2A receptor-negative cells in the somatosensory cortex in PACAP+/− mice compared with wild-type mice. These results indicate that PACAP+/− mice exhibit specific vulnerability to DOI-induced deficits in cortical sensory function, such as exaggerated head-twitch responses and sensorimotor gating deficits. Our findings provide insight into the neural mechanisms underlying impaired behavioral responses in which 5-HT2 receptors are implicated. PMID:24586556

  2. Csa-19, a radiation-responsive human gene, identified by an unbiased two-gel cDNA library screening method in human cancer cells

    NASA Technical Reports Server (NTRS)

    Balcer-Kubiczek, E. K.; Meltzer, S. J.; Han, L. H.; Zhang, X. F.; Shi, Z. M.; Harrison, G. H.; Abraham, J. M.

    1997-01-01

    A novel polymerase chain reaction (PCR)-based method was used to identify candidate genes whose expression is altered in cancer cells by ionizing radiation. Transcriptional induction of randomly selected genes in control versus irradiated human HL60 cells was compared. Among several complementary DNA (cDNA) clones recovered by this approach, one cDNA clone (CL68-5) was downregulated in X-irradiated HL60 cells but unaffected by 12-O-tetradecanoyl phorbol-13-acetate, forskolin, or cyclosporin-A. DNA sequencing of the CL68-5 cDNA revealed 100% nucleotide sequence homology to the reported human Csa-19 gene. Northern blot analysis of RNA from control and irradiated cells revealed the expression of a single 0.7-kilobase (kb) messenger RNA (mRNA) transcript. This 0.7-kb Csa-19 mRNA transcript was also expressed in a variety of human adult and corresponding fetal normal tissues. Moreover, when the effect of X- or fission neutron-irradiation on Csa-19 mRNA was compared in cultured human cells differing in p53 gene status (p53-/- versus p53+/+), downregulation of Csa-19 by X-rays or fission neutrons was similar in p53-wild type and p53-null cell lines. Our results provide the first known example of a radiation-responsive gene in human cancer cells whose expression is not associated with p53, adenylate cyclase or protein kinase C.

  3. Differential involvement of cyclase- versus non-cyclase-coupled D1-like dopamine receptors in orofacial movement topography in mice: studies with SKF 83822.

    PubMed

    Makihara, Yasuyuki; Okuda, Yaeko; Kawada, Chieko; Matsumoto, Mitsuhiko; Waddington, John L; Koshikawa, Noriaki; Tomiyama, Katsunori

    2007-03-19

    Though orofacial movements are fundamental motor patterns that are known to be regulated critically by D1-like dopamine receptors, these processes remain poorly understood. This uncertainty is heightened by evidence for putative D1-like receptors that are linked not only to adenylyl cyclase (AC) but also to phospholipase C (PLC). Using a new method, we have characterised four topographies of orofacial movement in the mouse using the novel D1-like agonist SKF 83822, which stimulates AC but not PLC. These were compared with responses to SKF 83959, which stimulates PLC but not AC. Also, effects were characterised using the D1-like antagonist SCH 23390 and the D2-like antagonist YM 09151-2. SKF 83822 induced vertical jaw movements with incisor chattering but inhibited horizontal jaw movements; there was little effect on tongue protrusions. Vertical jaw movements induced by SKF 83822 were inhibited by SCH 23390 but uninfluenced by YM 09151-2, while YM 09151-2 released horizontal jaw movements; thus, D1-like agonist-induced, AC-mediated vertical jaw movements constitute a 'pure' D1-like-dependent process that does not involve D1-like:D2-like interactions, while horizontal jaw movements involve oppositional interactions. Orofacial movements in mice appear to consist of at least four phenomenologically dissociable topographies that are mechanistically distinct. They are regulated differentially by AC- and/or PLC-dependent processes and these processes involve distinct D1-like:D2-like interactions.

  4. Sensor–response regulator interactions in a cross-regulated signal transduction network

    PubMed Central

    Huynh, TuAnh Ngoc; Chen, Li-Ling

    2015-01-01

    Two-component signal transduction involves phosphoryl transfer between a histidine kinase sensor and a response regulator effector. The nitrate-responsive two-component signal transduction systems in Escherichia coli represent a paradigm for a cross-regulation network, in which the paralogous sensor–response regulator pairs, NarX–NarL and NarQ–NarP, exhibit both cognate (e.g. NarX–NarL) and non-cognate (e.g. NarQ–NarL) interactions to control output. Here, we describe results from bacterial adenylate cyclase two-hybrid (BACTH) analysis to examine sensor dimerization as well as interaction between sensor–response regulator cognate and non-cognate pairs. Although results from BACTH analysis indicated that the NarX and NarQ sensors interact with each other, results from intragenic complementation tests demonstrate that they do not form functional heterodimers. Additionally, intragenic complementation shows that both NarX and NarQ undergo intermolecular autophosphorylation, deviating from the previously reported correlation between DHp (dimerization and histidyl phosphotransfer) domain loop handedness and autophosphorylation mode. Results from BACTH analysis revealed robust interactions for the NarX–NarL, NarQ–NarL and NarQ–NarP pairs but a much weaker interaction for the NarX–NarP pair. This demonstrates that asymmetrical cross-regulation results from differential binding affinities between different sensor–regulator pairs. Finally, results indicate that the NarL effector (DNA-binding) domain inhibits NarX–NarL interaction. Missense substitutions at receiver domain residue Ser-80 enhanced NarX–NarL interaction, apparently by destabilizing the NarL receiver–effector domain interface. PMID:25873583

  5. The cyclase-associated protein FgCap1 has both protein kinase A-dependent and -independent functions during deoxynivalenol production and plant infection in Fusarium graminearum.

    PubMed

    Yin, Tao; Zhang, Qiang; Wang, Jianhua; Liu, Huiquan; Wang, Chenfang; Xu, Jin-Rong; Jiang, Cong

    2017-01-31

    Fusarium graminearum is a causal agent of wheat scab and a producer of the trichothecene mycotoxin deoxynivalenol (DON). The expression of trichothecene biosynthesis (TRI) genes and DON production are mainly regulated by the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) pathway and two pathway-specific transcription factors (TRI6 and TRI10). Interestingly, deletion mutants of TRI6 show reduced expression of several components of cAMP signalling, including the FgCAP1 adenylate-binding protein gene that has not been functionally characterized in F. graminearum. In this study, we show that FgCap1 interacts with Fac1 adenylate cyclase and that deletion of FgCAP1 reduces the intracellular cAMP level and PKA activity. The Fgcap1 deletion mutant is defective in vegetative growth, conidiogenesis and plant infection. It also shows significantly reduced DON production and TRI gene expression, which can be suppressed by exogenous cAMP, indicating a PKA-dependent regulation of DON biosynthesis by FgCap1. The wild-type, but not tri6 mutant, shows increased levels of intracellular cAMP and FgCAP1 expression under DON-producing conditions. Furthermore, the promoter of FgCAP1 contains one putative Tri6-binding site that is important for its function during DON biosynthesis, but is dispensable for hyphal growth, conidiogenesis and pathogenesis. In addition, FgCap1 shows an actin-like localization to the cortical patches at the apical region of hyphal tips. Phosphorylation of FgCap1 at S353 was identified by phosphoproteomics analysis. The S353A mutation in FgCAP1 has no effect on its functions during vegetative growth, conidiation and DON production. However, expression of the FgCAP1(S353A) allele fails to complement the defects of the Fgcap1 mutant in plant infection, indicating the importance of the phosphorylation of FgCap1 at S353 during pathogenesis. Taken together, our results suggest that FgCAP1 is involved in the regulation of DON production via cAMP signalling

  6. Soluble Adenylyl Cyclase: Potential Role in Mineral Metabolism

    NASA Astrophysics Data System (ADS)

    Geng, Weidong; Moe, Orson W.

    2007-04-01

    The conversion of a signal of inorganic ion concentration to a second messenger is of fundamental biologic significance. The soluble adenylyl cyclase (sAC) serves such a purpose by transducing divalent cation concentrations and bicarbonate concentrations into intracellular cyclic AMP levels. sAC is widely expressed and may represent a generic bicarbonate sensor providing the afferent pathway to bicarbonate-regulated biologic processes. The best described action of sAC to date is in the mediation of bicarbonate-induced changes in sperm motility. Since a number of processes in mineral metabolism—such as bone formation, bone resorption, intestinal calcium absorption, and renal calcium transport in the distal convoluted tubule—are regulated by bicarbonate, sAC may play a role at multiple levels in mineral metabolism.

  7. Cloning, expression, and sequencing of squalene-hopene cyclase, a key enzyme in triterpenoid metabolism.

    PubMed Central

    Ochs, D; Kaletta, C; Entian, K D; Beck-Sickinger, A; Poralla, K

    1992-01-01

    The pentacyclic hopanoids, a class of eubacterial lipids, are synthesized by squalene-hopene cyclase and side chain-elongating enzymes. With the aid of DNA probes based on the amino-terminal sequence of purified squalene-hopene cyclase from Bacillus acidocaldarius, clones of Escherichia coli that express this enzyme in the cytoplasmic membrane were isolated. According to the DNA sequence, the cyclase contained 627 amino acids with a molecular mass of 69,473 Da. A high percentage of the amino acids were basic. No significant similarity to existing sequenced proteins was found. Images PMID:1729216

  8. On the dynamics of the adenylate energy system: homeorhesis vs homeostasis.

    PubMed

    De la Fuente, Ildefonso M; Cortés, Jesús M; Valero, Edelmira; Desroches, Mathieu; Rodrigues, Serafim; Malaina, Iker; Martínez, Luis

    2014-01-01

    Biochemical energy is the fundamental element that maintains both the adequate turnover of the biomolecular structures and the functional metabolic viability of unicellular organisms. The levels of ATP, ADP and AMP reflect roughly the energetic status of the cell, and a precise ratio relating them was proposed by Atkinson as the adenylate energy charge (AEC). Under growth-phase conditions, cells maintain the AEC within narrow physiological values, despite extremely large fluctuations in the adenine nucleotides concentration. Intensive experimental studies have shown that these AEC values are preserved in a wide variety of organisms, both eukaryotes and prokaryotes. Here, to understand some of the functional elements involved in the cellular energy status, we present a computational model conformed by some key essential parts of the adenylate energy system. Specifically, we have considered (I) the main synthesis process of ATP from ADP, (II) the main catalyzed phosphotransfer reaction for interconversion of ATP, ADP and AMP, (III) the enzymatic hydrolysis of ATP yielding ADP, and (IV) the enzymatic hydrolysis of ATP providing AMP. This leads to a dynamic metabolic model (with the form of a delayed differential system) in which the enzymatic rate equations and all the physiological kinetic parameters have been explicitly considered and experimentally tested in vitro. Our central hypothesis is that cells are characterized by changing energy dynamics (homeorhesis). The results show that the AEC presents stable transitions between steady states and periodic oscillations and, in agreement with experimental data these oscillations range within the narrow AEC window. Furthermore, the model shows sustained oscillations in the Gibbs free energy and in the total nucleotide pool. The present study provides a step forward towards the understanding of the fundamental principles and quantitative laws governing the adenylate energy system, which is a fundamental element for

  9. On the Dynamics of the Adenylate Energy System: Homeorhesis vs Homeostasis

    PubMed Central

    De la Fuente, Ildefonso M.; Cortés, Jesús M.; Valero, Edelmira; Desroches, Mathieu; Rodrigues, Serafim; Malaina, Iker; Martínez, Luis

    2014-01-01

    Biochemical energy is the fundamental element that maintains both the adequate turnover of the biomolecular structures and the functional metabolic viability of unicellular organisms. The levels of ATP, ADP and AMP reflect roughly the energetic status of the cell, and a precise ratio relating them was proposed by Atkinson as the adenylate energy charge (AEC). Under growth-phase conditions, cells maintain the AEC within narrow physiological values, despite extremely large fluctuations in the adenine nucleotides concentration. Intensive experimental studies have shown that these AEC values are preserved in a wide variety of organisms, both eukaryotes and prokaryotes. Here, to understand some of the functional elements involved in the cellular energy status, we present a computational model conformed by some key essential parts of the adenylate energy system. Specifically, we have considered (I) the main synthesis process of ATP from ADP, (II) the main catalyzed phosphotransfer reaction for interconversion of ATP, ADP and AMP, (III) the enzymatic hydrolysis of ATP yielding ADP, and (IV) the enzymatic hydrolysis of ATP providing AMP. This leads to a dynamic metabolic model (with the form of a delayed differential system) in which the enzymatic rate equations and all the physiological kinetic parameters have been explicitly considered and experimentally tested in vitro. Our central hypothesis is that cells are characterized by changing energy dynamics (homeorhesis). The results show that the AEC presents stable transitions between steady states and periodic oscillations and, in agreement with experimental data these oscillations range within the narrow AEC window. Furthermore, the model shows sustained oscillations in the Gibbs free energy and in the total nucleotide pool. The present study provides a step forward towards the understanding of the fundamental principles and quantitative laws governing the adenylate energy system, which is a fundamental element for

  10. Adenylyl cyclase 2 selectively couples to E prostanoid type 2 receptors, whereas adenylyl cyclase 3 is not receptor-regulated in airway smooth muscle.

    PubMed

    Bogard, Amy S; Adris, Piyatilake; Ostrom, Rennolds S

    2012-08-01

    Adenylyl cyclases (ACs) are important regulators of airway smooth muscle function, because β-adrenergic receptor (βAR) agonists stimulate AC activity and cAMP production. We have previously shown in a number of cell types that AC6 selectively couples to βAR and these proteins are coexpressed in lipid rafts. We overexpressed AC2, AC3, and AC6 in mouse bronchial smooth muscle cells (mBSMCs) and human embryonic kidney (HEK)-293 cells by using recombinant adenoviruses and assessed their localization and regulation by various G protein-coupled receptors (GPCRs). AC3 and AC6 were expressed primarily in caveolin-rich fractions, whereas AC2 expression was excluded from these domains. AC6 expression enhanced cAMP production in response to isoproterenol but did not increase responses to butaprost, reflecting the colocalization of AC6 with β(2)AR but not E prostanoid type 2 receptor (EP(2)R) in lipid raft fractions. AC2 expression enhanced butaprost-stimulated cAMP production but had no effect on the β(2)AR-mediated response. AC3 did not couple to any GPCR tested. Forskolin-induced arborization of mBSMCs was assessed as a functional readout of cAMP signaling. Arborization was enhanced by overexpression of AC6 and AC3, but AC2 had no effect. GPCR-stimulated arborization mirrored the selective coupling observed for cAMP production. With the addition of the phosphodiesterase 4 (PDE4) inhibitor rolipram AC2 accelerated forskolin-stimulated arborization. Thus, AC2 selectively couples to EP(2)R, but signals from this complex are limited by PDE4 activity. AC3 does not seem to couple to GPCR in either mBSMCs or HEK-293 cells, so it probably exists in a distinct signaling domain in these cells.

  11. Diadenosine Homodinucleotide Products of ADP-ribosyl Cyclases Behave as Modulators of the Purinergic Receptor P2X7*

    PubMed Central

    Bruzzone, Santina; Basile, Giovanna; Chothi, Madhu Parakkottil; Nobbio, Lucilla; Usai, Cesare; Jacchetti, Emanuela; Schenone, Angelo; Guse, Andreas H.; Di Virgilio, Francesco; De Flora, Antonio; Zocchi, Elena

    2010-01-01

    ADP-ribosyl cyclases from both vertebrates and invertebrates were previously shown to produce two isomers of P1,P2 diadenosine 5′,5′"-P1, P2-diphosphate, P18 and P24, from cyclic ADP-ribose (cADPR) and adenine. P18 and P24 are characterized by an unusual N-glycosidic linkage in one of the adenylic mononucleotides (Basile, G., Taglialatela-Scafati, O., Damonte, G., Armirotti, A., Bruzzone, S., Guida, L., Franco, L., Usai, C., Fattorusso, E., De Flora, A., and Zocchi, E. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 14509–14514). P24, but not P18, proved to increase the intracellular Ca2+ concentration ([Ca2+]i) in HeLa cells and to negatively affect mitochondrial function. Here we show that micromolar P24, but not P18, triggers a slow and sustained influx of extracellular Ca2+ through the opening of the purinergic receptor/channel P2X7. On the other hand, P18 inhibits the Ca2+ influx induced by 0.6 mm ATP in HEK293 cells stably transfected with P2X7, with an IC50 of ∼1 μm. Thus, P18 is devoid of intrinsic P2X7 stimulatory activity and behaves as an ATP antagonist. A P2X7-mediated increase of the basal [Ca2+]i has been demonstrated to negatively affect Schwann cell (SC) function in rats with the inherited, peripheral neuropathy Charcot-Marie-Tooth 1A (CMT1A) (Nobbio, L., Sturla, L., Fiorese, F., Usai, C., Basile, G., Moreschi, I., Benvenuto, F., Zocchi, E., De Flora, A., Schenone, A., and Bruzzone S. (2009) J. Biol. Chem. 284, 23146–23158). Preincubation of CMT1A SC with 200 nm P18 restored the basal [Ca2+]i to values similar to those recorded in wild-type SC. These results identify P18 as a new P2X7 antagonist, potentially useful in the treatment of CMT1A. PMID:20439466

  12. Culture and characteristics of hormone-responsive neuroblastoma x glioma hybrid cells

    SciTech Connect

    Hamprecht, B.; Glaser, T.; Reiser, G.; Bayer, E.; Propst, F.

    1985-01-01

    Neuroblastoma x glioma hybrid cells were generated by cell fusion of the 6-thioguanine-resistant clonal mouse neuroblastoma cells and the bromodeoxyuridine-resistant rat glioma cells, selection, and cloning. Every characteristics generally ascribed to neurons has been observed with the hybrid cells. The paper explores the morphological differentiation of hybrid cells, procedures for testing the hormonal regulation of intracellular levels of cyclic, (/sup 3/H)AMP in hybrid cells, hormonal regulation of adenylate cyclase in homogenates of hyrbid cells, intracellular levels of cyclic GMP, and uptake of guanidinium ions in hybrid cells.

  13. NMR studies of the AMP-binding site and mechanism of adenylate kinase.

    PubMed

    Fry, D C; Kuby, S A; Mildvan, A S

    1987-03-24

    NMR has previously been used to determine the conformation of enzyme-bound MgATP and to locate the MgATP-binding site on adenylate kinase [Fry, D. C., Kuby, S. A., & Mildvan, A. S. (1985) Biochemistry 24, 4680-4694]. To determine the conformation and location of the other substrate, AMP, distances have been measured from Cr3+AMPPCP, a linear competitive inhibitor with respect to MgATP, to six protons and to the phosphorus atom of AMP on adenylate kinase, with the paramagnetic probe-T1 method. Time-dependent nuclear Overhauser effects (NOEs) have been used to measure five interproton distances on enzyme-bound AMP. These distances were used to determine the conformation of bound AMP in addition to its position with respect to metal-ATP. Enzyme-bound AMP exhibits a high anti-glycosyl torsional angle (chi = 110 +/- 10 degrees), a 3'-endo,2'-exo ribose pucker (delta = 105 +/- 10 degrees), and gauche-trans orientations about the C4'-C5' bond (gamma = 180 +/- 10 degrees) and the C5'-O5' bond (beta = 170 +/- 20 degrees). The distance from Cr3+ to the phosphorus of AMP is 5.9 +/- 0.3 A, indicating a reaction coordinate distance of approximately 3 A, which is consistent with an associative SN2 mechanism for the phosphoryl transfer. Ten intermolecular NOEs, from protons of the enzyme to those of AMP, were detected, indicating the proximity of at least three hydrophobic amino acids to bound AMP. These constraints, together with the conformation of AMP and the intersubstrate distances, were used to position AMP into the X-ray structure of adenylate kinase. The AMP binding site is found to be near (less than or equal to 4 A from) Leu-116, Arg-171, Val-173, Val-182, and Leu-190; all of these residues have been found to be invariant in muscle-type rabbit, calf, human, porcine [Kuby, S. A., Palmieri, R. H., Frischat, A., Fischer, A. H., Wu, L. H., Maland, L., & Manship, M. (1984) Biochemistry 23, 2393-2399], and chicken adenylate kinase [Kishi, F., Maruyama, M., Tanizawa, Y

  14. Crystal Structures of the Adenylate Sensor from Fission Yeast AMP-Activated Protein Kinase

    SciTech Connect

    Townley,R.; Shapiro, L.

    2007-01-01

    The 5'-AMP (adenosine monophosphate)-activated protein kinase (AMPK) coordinates metabolic function with energy availability by responding to changes in intracellular adenosine triphosphate (ATP) and AMP levels. Here we report crystal structures at 2.6 and 2.9 Angstrom resolution for ATP- and AMP-bound forms of a core {alpha}{beta}{gamma} adenylate-binding domain from the fission yeast AMPK homologue. ATP and AMP bind competitively to a single site in the {gamma} subunit, with their respective phosphate groups positioned near function-impairing mutants. Surprisingly, ATP binds without counter ions, amplifying its electrostatic effects on a critical regulatory region where all three subunits converge.

  15. Hydroxamate-based colorimetric assay to assess amide bond formation by adenylation domain of nonribosomal peptide synthetases.

    PubMed

    Hara, Ryotaro; Suzuki, Ryohei; Kino, Kuniki

    2015-05-15

    We demonstrated the usefulness of a hydroxamate-based colorimetric assay for predicting amide bond formation (through an aminoacyl-AMP intermediate) by the adenylation domain of nonribosomal peptide synthetases. By using a typical adenylation domain of tyrocidine synthetase (involved in tyrocidine biosynthesis), we confirmed the correlation between the absorbance at 490 nm of the l-Trp-hydroxamate-Fe(3+) complex and the formation of l-Trp-l-Pro, where l-Pro was used instead of hydroxylamine. Furthermore, this assay was adapted to the adenylation domains of surfactin synthetase (involved in surfactin biosynthesis) and bacitracin synthetase (involved in bacitracin biosynthesis). Consequently, the formation of various aminoacyl l-Pro formations was observed.

  16. Response of the corpus luteum to luteinizing hormone.

    PubMed Central

    Niswender, G D

    1981-01-01

    The response of steroidogenic tissues to tropic hormones is regulated in part by specific receptors in the target cells for the stimulatory hormone. As a result of hormone binding to receptor the enzyme adenylate cyclase is activated with a resultant increase in intracellular levels of cAMP. Enhanced protein kinase activity then leads to increased steroidogenesis via several possible mechanisms, including direct activation of components of steroidogenic enzyme systems via phosphorylation. The initial effects of tropic hormones such as LH are dependent upon the number of receptors present on the surface of the target cell. Numerous factors influence the number of LH receptors in the corpus luteum. A model is presented for the mechanisms involved in the loss and renewal of LH receptors in the luteal cell. The life of the LH receptor on luteal cells appears to be a single binding of hormone. The hormone-receptor complex is then internalized by endocytosis and the hormone is degraded in lysosomes. After internalization the receptor is also degraded in lysosomes or recycled via the Golgi apparatus. New or recycled receptors for LH are incorporated into the limiting membrane of protein containing secretory granules. One of the actions of LH is enhancement of the exocytosis of these secretory granules with incorporation of the limiting membrane (and the LH receptors?) of the granule into the plasma membrane of the cell. These proposed mechanisms explain the increase in the number of receptors for LH seen immediately after stimulation of the luteal cell with massive doses of LH and also explain the "down-regulation" of LH receptors 24 hr after administration of LH. PMID:6263609

  17. Terpenoid biosynthesis off the beaten track: unconventional cyclases and their impact on biomimetic synthesis.

    PubMed

    Baunach, Martin; Franke, Jakob; Hertweck, Christian

    2015-02-23

    Terpene and terpenoid cyclizations are counted among the most complex chemical reactions occurring in nature and contribute crucially to the tremendous structural diversity of this largest family of natural products. Many studies were conducted at the chemical, genetic, and biochemical levels to gain mechanistic insights into these intriguing reactions that are catalyzed by terpene and terpenoid cyclases. A myriad of these enzymes have been characterized. Classical textbook knowledge divides terpene/terpenoid cyclases into two major classes according to their structure and reaction mechanism. However, recent discoveries of novel types of terpenoid cyclases illustrate that nature's enzymatic repertoire is far more diverse than initially thought. This Review outlines novel terpenoid cyclases that are out of the ordinary.

  18. Bicarbonate-sensing soluble adenylyl cyclase is present in the cell cytoplasm and nucleus of multiple shark tissues.

    PubMed

    Roa, Jinae N; Tresguerres, Martin

    2017-01-01

    The enzyme soluble adenylyl cyclase (sAC) is directly stimulated by bicarbonate (HCO3(-)) to produce the signaling molecule cyclic adenosine monophosphate (cAMP). Because sAC and sAC-related enzymes are found throughout phyla from cyanobacteria to mammals and they regulate cell physiology in response to internal and external changes in pH, CO2, and HCO3(-), sAC is deemed an evolutionarily conserved acid-base sensor. Previously, sAC has been reported in dogfish shark and round ray gill cells, where they sense and counteract blood alkalosis by regulating the activity of V-type H(+)- ATPase. Here, we report the presence of sAC protein in gill, rectal gland, cornea, intestine, white muscle, and heart of leopard shark Triakis semifasciata Co-expression of sAC with transmembrane adenylyl cyclases supports the presence of cAMP signaling microdomains. Furthermore, immunohistochemistry on tissue sections, and western blots and cAMP-activity assays on nucleus-enriched fractions demonstrate the presence of sAC protein in and around nuclei. These results suggest that sAC modulates multiple physiological processes in shark cells, including nuclear functions.

  19. Isolation and functional characterization of a lycopene beta-cyclase gene that controls fruit colour of papaya (Carica papaya L.).

    PubMed

    Devitt, Luke C; Fanning, Kent; Dietzgen, Ralf G; Holton, Timothy A

    2010-01-01

    The colour of papaya fruit flesh is determined largely by the presence of carotenoid pigments. Red-fleshed papaya fruit contain lycopene, whilst this pigment is absent from yellow-fleshed fruit. The conversion of lycopene (red) to beta-carotene (yellow) is catalysed by lycopene beta-cyclase. This present study describes the cloning and functional characterization of two different genes encoding lycopene beta-cyclases (lcy-beta1 and lcy-beta2) from red (Tainung) and yellow (Hybrid 1B) papaya cultivars. A mutation in the lcy-beta2 gene, which inactivates enzyme activity, controls lycopene production in fruit and is responsible for the difference in carotenoid production between red and yellow-fleshed papaya fruit. The expression level of both lcy-beta1 and lcy-beta2 genes is similar and low in leaves, but lcy-beta2 expression increases markedly in ripe fruit. Isolation of the lcy-beta2 gene from papaya, that is preferentially expressed in fruit and is correlated with fruit colour, will facilitate marker-assisted breeding for fruit colour in papaya and should create possibilities for metabolic engineering of carotenoid production in papaya fruit to alter both colour and nutritional properties.

  20. Identification of the silkworm quail gene reveals a crucial role of a receptor guanylyl cyclase in larval pigmentation.

    PubMed

    Yuasa, Masashi; Kiuchi, Takashi; Banno, Yutaka; Katsuma, Susumu; Shimada, Toru

    2016-01-01

    Diverse color patterns on the integument of lepidopteran larvae play important roles in their survival through camouflage, mimicry, sexual signaling, and aposematism. In the silkworm Bombyx mori, many color pattern variations have been preserved in inbred strains making them a good model for elucidating the molecular mechanisms that underlie color pattern formation. In this study, we focused on the silkworm quail (q) mutant, which exhibits abnormalities in multiple pigment biosynthesis pathways. Positional cloning of the q gene revealed that disruption of a guanylyl cyclase gene, BmGC-I, is responsible for its abnormal pigmentation. In q mutants, we identified a 16-bp deletion in the BmGC-I transcript, resulting in the production of a premature stop codon. Knockout of the BmGC-I gene resulted in the q-like abnormal pigmentation, thereby demonstrating that the BmGC-I gene is involved in the pigment biosynthesis pathway in the integument. Moreover, quantitative reverse transcription polymerase chain reaction showed that BmGC-I was strongly expressed in the fourth instar on day 2. Our results suggest that BmGC-I deficiency affects the pigment biosynthesis pathway, which supports the involvement of guanylyl cyclase in larval coloration.

  1. 2'-Phosphate cyclase activity of RtcA: a potential rationale for the operon organization of RtcA with an RNA repair ligase RtcB in Escherichia coli and other bacterial taxa.

    PubMed

    Das, Ushati; Shuman, Stewart

    2013-10-01

    RNA terminal phosphate cyclase catalyzes the ATP-dependent conversion of a 3'-phosphate RNA end to a 2',3'-cyclic phosphate via covalent enzyme-(histidinyl-Nε)-AMP and RNA(3')pp(5')A intermediates. Here, we report that Escherichia coli RtcA (and its human homolog Rtc1) are capable of cyclizing a 2'-phosphate RNA end in high yield. The rate of 2'-phosphate cyclization by RtcA is five orders of magnitude slower than 3'-phosphate cyclization, notwithstanding that RtcA binds with similar affinity to RNA3'p and RNA2'p substrates. These findings expand the functional repertoire of RNA cyclase and suggest that phosphate geometry during adenylate transfer to RNA is a major factor in the kinetics of cyclization. RtcA is coregulated in an operon with an RNA ligase, RtcB, that splices RNA 5'-OH ends to either 3'-phosphate or 2',3'-cyclic phosphate ends. Our results suggest that RtcA might serve an end healing function in an RNA repair pathway, by converting RNA 2'-phosphates, which cannot be spliced by RtcB, to 2',3'-cyclic phosphates that can be sealed. The rtcBA operon is controlled by the σ(54) coactivator RtcR encoded by an adjacent gene. This operon arrangement is conserved in diverse bacterial taxa, many of which have also incorporated the RNA-binding protein Ro (which is implicated in RNA quality control under stress conditions) as a coregulated component of the operon.

  2. Choreographing the adenylyl cyclase signalosome: sorting out the partners and the steps.

    PubMed

    Ostrom, Rennolds S; Bogard, Amy S; Gros, Robert; Feldman, Ross D

    2012-01-01

    Adenylyl cyclases are a ubiquitous family of enzymes and are critical regulators of metabolic and cardiovascular function. Multiple isoforms of the enzyme are expressed in a range of tissues. However, for many processes, the adenylyl cyclase isoforms have been thought of as essentially interchangeable, with their impact more dependent on their common actions to increase intracellular cyclic adenosine monophosphate content regardless of the isoform involved. It has long been appreciated that each subfamily of isoforms demonstrate a specific pattern of "upstream" regulation, i.e., specific patterns of ion dependence (e.g., calcium-dependence) and specific patterns of regulation by kinases (protein kinase A (PKA), protein kinase C (PKC), raf). However, more recent studies have suggested that adenylyl cyclase isoform-selective patterns of signaling are a wide-spread phenomenon. The determinants of these selective signaling patterns relate to a number of factors, including: (1) selective coupling of specific adenylyl cyclase isoforms with specific G protein-coupled receptors, (2) localization of specific adenylyl cyclase isoforms in defined structural domains (AKAP complexes, caveolin/lipid rafts), and (3) selective coupling of adenylyl cyclase isoforms with specific downstream signaling cascades important in regulation of cell growth and contractility. The importance of isoform-specific regulation has now been demonstrated both in mouse models as well as in humans. Adenylyl cyclase has not been viewed as a useful target for therapeutic regulation, given the ubiquitous expression of the enzyme and the perceived high risk of off-target effects. Understanding which isoforms of adenylyl cyclase mediate distinct cellular effects would bring new significance to the development of isoform-specific ligands to regulate discrete cellular actions.

  3. [Soluble guanylate cyclase in the molecular mechanism underlying the therapeutic action of drugs].

    PubMed

    Piatakova, N V; Severina, I S

    2012-01-01

    The influence of ambroxol--a mucolytic drug--on the activity of human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase and activation of both enzymes by NO-donors (sodium nitroprusside and Sin-1) were investigated. Ambroxol in the concentration range from 0.1 to 10 microM had no effect on the basal activity of both enzymes. Ambroxol inhibited in a concentration-dependent manner the sodium nitroprusside-induced human platelet soluble guanylate cyclase and rat lung soluble guanylate cyclase with the IC50 values 3.9 and 2.1 microM, respectively. Ambroxol did not influence the stimulation of both enzymes by protoporphyrin IX. The influence of artemisinin--an antimalarial drug--on human platelet soluble guanylate cyclase activity and the enzyme activation by NO-donors were investigated. Artemisinin (0.1-100 microM) had no effect on the basal activity of the enzyme. Artemisinin inhibited in a concentration-dependent manner the sodium nitroprusside-induced activation of human platelet guanylate cyclase with an IC50 value 5.6 microM. Artemisinin (10 microM) also inhibited (by 71 +/- 4.0%) the activation of the enzyme by thiol-dependent NO-donor the derivative of furoxan, 3,4-dicyano-1,2,5-oxadiazolo-2-oxide (10 microM), but did not influence the stimulation of soluble guanylate cyclase by protoporphyrin IX. It was concluded that the sygnalling system NO-soluble guanylate cyclase-cGMP is involved in the molecular mechanism of the therapeutic action of ambroxol and artemisinin.

  4. Receptor guanylyl cyclase-G is a novel thermosensory protein activated by cool temperatures.

    PubMed

    Chao, Ying-Chi; Chen, Chih-Cheng; Lin, Yuh-Charn; Breer, Heinz; Fleischer, Joerg; Yang, Ruey-Bing

    2015-02-03

    Transmembrane guanylyl cyclases (GCs), with activity regulated by peptide ligands and/or calcium-binding proteins, are essential for various physiological and sensory processes. The mode of activation of the GC subtype GC-G, which is expressed in neurons of the Grueneberg ganglion that respond to cool temperatures, has been elusive. In searching for appropriate stimuli to activate GC-G, we found that its enzymatic activity is directly stimulated by cool temperatures. In this context, it was observed that dimerization/oligomerization of GC-G, a process generally considered as critical for enzymatic activity of GCs, is strongly enhanced by coolness. Moreover, heterologous expression of GC-G in cultured cells rendered these cells responsive to coolness; thus, the protein might be a sensor for cool temperatures. This concept is supported by the observation of substantially reduced coolness-induced response of Grueneberg ganglion neurons and coolness-evoked ultrasonic vocalization in GC-G-deficient mouse pups. GC-G may be a novel thermosensory protein with functional implications for the Grueneberg ganglion, a sensory organ responding to cool temperatures.

  5. Drug Target Exploitable Structural Features of Adenylyl Cyclase Activity in Schistosoma mansoni

    PubMed Central

    Mbah, Andreas N.; Kamga, Henri L.; Awofolu, Omotayo R.; Isokpehi, Raphael D.

    2012-01-01

    The draft genome sequence of the parasitic flatworm Schistosoma mansoni (S. mansoni), a cause of schistosomiasis, encodes a predicted guanosine triphosphate (GTP) binding protein tagged Smp_059340.1. Smp_059340.1 is predicted to be a member of the G protein alpha-s subunit responsible for regulating adenylyl cyclase activity in S. mansoni and a possible drug target against the parasite. Our structural bioinformatics analyses identified key amino acid residues (Ser53, Thr188, Asp207 and Gly210) in the two molecular switches responsible for cycling the protein between active (GTP bound) and inactive (GDP bound) states. Residue Thr188 is located on Switch I region while Gly210 is located on Switch II region with Switch II longer than Switch I. The Asp207 is located on the G3 box motif and Ser53 is the binding residue for magnesium ion. These findings offer new insights into the dynamic and functional determinants of the Smp_059340.1 protein in regulating the S. mansoni life cycle. The binding interfaces and their residues could be used as starting points for selective modulations of interactions within the pathway using small molecules, peptides or mutagenesis. PMID:23133313

  6. Opening mechanism of adenylate kinase can vary according to selected molecular dynamics force field.

    PubMed

    Unan, Hulya; Yildirim, Ahmet; Tekpinar, Mustafa

    2015-07-01

    Adenylate kinase is a widely used test case for many conformational transition studies. It performs a large conformational transition between closed and open conformations while performing its catalytic function. To understand conformational transition mechanism and impact of force field choice on E. Coli adenylate kinase, we performed all-atom explicit solvent classical molecular dynamics simulations starting from the closed conformation with four commonly used force fields, namely, Amber99, Charmm27, Gromos53a6, Opls-aa. We carried out 40 simulations, each one 200 ns. We analyzed completely 12 of them that show full conformational transition from the closed state to the open one. Our study shows that different force fields can have a bias toward different transition pathways. Transition time scales, frequency of conformational transitions, order of domain motions and free energy landscapes of each force field may also vary. In general, Amber99 and Charmm27 behave similarly while Gromos53a6 results have a resemblance to the Opls-aa force field results.

  7. Design, synthesis, and biological evaluation of α-hydroxyacyl-AMS inhibitors of amino acid adenylation enzymes.

    PubMed

    Davis, Tony D; Mohandas, Poornima; Chiriac, Maria I; Bythrow, Glennon V; Quadri, Luis E N; Tan, Derek S

    2016-11-01

    Biosynthesis of bacterial natural-product virulence factors is emerging as a promising antibiotic target. Many such natural products are produced by nonribosomal peptide synthetases (NRPS) from amino acid precursors. To develop selective inhibitors of these pathways, we have previously described aminoacyl-AMS (sulfamoyladenosine) macrocycles that inhibit NRPS amino acid adenylation domains but not mechanistically-related aminoacyl-tRNA synthetases. To improve the cell permeability of these inhibitors, we explore herein replacement of the α-amino group with an α-hydroxy group. In both macrocycles and corresponding linear congeners, this leads to decreased biochemical inhibition of the cysteine adenylation domain of the Yersina pestis siderophore synthetase HMWP2, which we attribute to loss of an electrostatic interaction with a conserved active-site aspartate. However, inhibitory activity can be regained by installing a cognate β-thiol moiety in the linear series. This provides a path forward to develop selective, cell-penetrant inhibitors of the biosynthesis of virulence factors to probe their biological functions and potential as therapeutic targets.

  8. Study into the kinetic properties and surface attachment of a thermostable adenylate kinase

    PubMed Central

    Hathaway, H.J.; Sutton, J.M.; Jenkins, A.T.A.

    2015-01-01

    A thermostable adenylate kinase (tAK) has been used as model protein contaminant on surfaces, so used because residual protein after high temperature wash steps can be detected at extremely low concentrations. This gives the potential for accurate, quantitative measurement of the effectiveness of different wash processes in removing protein contamination. Current methods utilise non-covalent (physisorbtion) of tAK to surfaces, but this can be relatively easily removed. In this study, the covalent binding of tAK to surfaces was studied to provide an alternative model for surface contamination. Kinetic analysis showed that the efficiency of the enzyme expressed as the catalytic rate over the Michaelis constant (kcat/KM) increased from 8.45±3.04 mM−1 s−1 in solution to 32.23±3.20 or 24.46±4.41 mM−1 s−1 when the enzyme was immobilised onto polypropylene or plasma activated polypropylene respectively. Maleic anhydride plasma activated polypropylene showed potential to provide a more robust challenge for washing processes as it retained significantly higher amounts of tAK enzyme than polypropylene in simple washing experiments. Inhibition of the coupled enzyme (luciferase/luciferin) system used for the detection of adenylate kinase activity, was observed for a secondary product of the reaction. This needs to be taken into consideration when using the assay to estimate cleaning efficacy. PMID:26339684

  9. Leveraging the Mechanism of Oxidative Decay for Adenylate Kinase to Design Structural and Functional Resistances

    PubMed Central

    Howell, Stanley C.; Richards, David H.; Mitch, William A.; Wilson, Corey J.

    2016-01-01

    Characterization of the mechanisms underlying hypohalous acid (i.e., hypochlorous acid or hypobromous acid) degradation of proteins is important for understanding how the immune system deactivates pathogens during infections, and damages human tissues during inflammatory diseases. Proteins are particularly important hypohalous acid reaction targets in pathogens and in host tissues, as evidenced by the detection of chlorinated and brominated oxidizable residues. While a significant amount of work has been conducted for reactions of hypohalous acids with a range of individual amino acids and small peptides, the assessment of oxidative decay in full-length proteins has lagged in comparison. The most rigorous test of our understanding of oxidative decay of proteins is the rational redesign of proteins with conferred resistances to the decay of structure and function. Toward this end, in this study we experimentally determined a putative mechanism of oxidative decay using adenylate kinase as the model system. In turn, we leveraged this mechanism to rationally design new proteins and experimentally test each system for oxidative resistance to loss of structure and function. From our extensive assessment of secondary-structure, protein hydrodynamics and enzyme activity upon hypochlorous acid or hypobromous acid challenge, we have identified two key strategies for conferring structural and functional resistance. Namely, the design of proteins (adenylate kinase enzymes) that are resistant to oxidation requires complementary consideration of protein stability and the modification (elimination) of certain oxidizable residues proximal to catalytic sites. PMID:26266833

  10. Opening mechanism of adenylate kinase can vary according to selected molecular dynamics force field

    NASA Astrophysics Data System (ADS)

    Unan, Hulya; Yildirim, Ahmet; Tekpinar, Mustafa

    2015-07-01

    Adenylate kinase is a widely used test case for many conformational transition studies. It performs a large conformational transition between closed and open conformations while performing its catalytic function. To understand conformational transition mechanism and impact of force field choice on E. Coli adenylate kinase, we performed all-atom explicit solvent classical molecular dynamics simulations starting from the closed conformation with four commonly used force fields, namely, Amber99, Charmm27, Gromos53a6, Opls-aa. We carried out 40 simulations, each one 200 ns. We analyzed completely 12 of them that show full conformational transition from the closed state to the open one. Our study shows that different force fields can have a bias toward different transition pathways. Transition time scales, frequency of conformational transitions, order of domain motions and free energy landscapes of each force field may also vary. In general, Amber99 and Charmm27 behave similarly while Gromos53a6 results have a resemblance to the Opls-aa force field results.

  11. Structure of the adenylation domain of NAD[superscript +]-dependent DNA ligase from Staphylococcus aureus

    SciTech Connect

    Han, Seungil; Chang, Jeanne S.; Griffor, Matt; Pfizer

    2010-09-17

    DNA ligase catalyzes phosphodiester-bond formation between immediately adjacent 5'-phosphate and 3''-hydroxyl groups in double-stranded DNA and plays a central role in many cellular and biochemical processes, including DNA replication, repair and recombination. Bacterial NAD{sup +}-dependent DNA ligases have been extensively characterized as potential antibacterial targets because of their essentiality and their structural distinction from human ATP-dependent DNA ligases. The high-resolution structure of the adenylation domain of Staphylococcus aureus NAD{sup +}-dependent DNA ligase establishes the conserved domain architecture with other bacterial adenylation domains. Two apo crystal structures revealed that the active site possesses the preformed NAD{sup +}-binding pocket and the 'C2 tunnel' lined with hydrophobic residues: Leu80, Phe224, Leu287, Phe295 and Trp302. The C2 tunnel is unique to bacterial DNA ligases and the Leu80 side chain at the mouth of the tunnel points inside the tunnel and forms a narrow funnel in the S. aureus DNA ligase structure. Taken together with other DNA ligase structures, the S. aureus DNA ligase structure provides a basis for a more integrated understanding of substrate recognition and catalysis and will be also be of help in the development of small-molecule inhibitors.

  12. Purine and pyrimidine nucleosides preserve human astrocytoma cell adenylate energy charge under ischemic conditions.

    PubMed

    Balestri, Francesco; Giannecchini, Michela; Sgarrella, Francesco; Carta, Maria Caterina; Tozzi, Maria Grazia; Camici, Marcella

    2007-02-01

    The brain depends on both glycolysis and mitochondrial oxidative phosphorylation for maintenance of ATP pools. Astrocytes play an integral role in brain functions providing trophic supports and energy substrates for neurons. In this paper, we report that human astrocytoma cells (ADF) undergoing ischemic conditions may use both purine and pyrimidine nucleosides as energy source to slow down cellular damage. The cells are subjected to metabolic stress conditions by exclusion of glucose and incubation with oligomycin (an inhibitor of oxidative phosphorylation). This treatment brings about a depletion of the ATP pool, with a concomitant increase in the AMP levels, which results in a significant decrease of the adenylate energy charge. The presence of purine nucleosides in the culture medium preserves the adenylate energy charge, and improves cell viability. Besides purine nucleosides, also pyrimidine nucleosides, such as uridine and, to a lesser extent, cytidine, are able to preserve the ATP pool. The determination of lactate in the incubation medium indicates that nucleosides can preserve the ATP pool through anaerobic glycolysis, thus pointing to a relevant role of the phosphorolytic cleavage of the N-glycosidic bond of nucleosides which generates, without energy expense, the phosphorylated pentose, which through the pentose phosphate pathway and glycolysis can be converted to energetic intermediates also in the absence of oxygen. In fact, ADF cells possess both purine nucleoside phosphorylase and uridine phosphorylase activities.

  13. Leveraging the Mechanism of Oxidative Decay for Adenylate Kinase to Design Structural and Functional Resistances.

    PubMed

    Howell, Stanley C; Richards, David H; Mitch, William A; Wilson, Corey J

    2015-10-16

    Characterization of the mechanisms underlying hypohalous acid (i.e., hypochlorous acid or hypobromous acid) degradation of proteins is important for understanding how the immune system deactivates pathogens during infections and damages human tissues during inflammatory diseases. Proteins are particularly important hypohalous acid reaction targets in pathogens and in host tissues, as evidenced by the detection of chlorinated and brominated oxidizable residues. While a significant amount of work has been conducted for reactions of hypohalous acids with a range of individual amino acids and small peptides, the assessment of oxidative decay in full-length proteins has lagged in comparison. The most rigorous test of our understanding of oxidative decay of proteins is the rational redesign of proteins with conferred resistances to the decay of structure and function. Toward this end, in this study, we experimentally determined a putative mechanism of oxidative decay using adenylate kinase as the model system. In turn, we leveraged this mechanism to rationally design new proteins and experimentally test each system for oxidative resistance to loss of structure and function. From our extensive assessment of secondary structure, protein hydrodynamics, and enzyme activity upon hypochlorous acid or hypobromous acid challenge, we have identified two key strategies for conferring structural and functional resistance, namely, the design of proteins (adenylate kinase enzymes) that are resistant to oxidation requires complementary consideration of protein stability and the modification (elimination) of certain oxidizable residues proximal to catalytic sites.

  14. Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120

    PubMed Central

    Frébortová, Jitka; Greplová, Marta; Seidl, Michael F.; Heyl, Alexander; Frébort, Ivo

    2015-01-01

    Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants. PMID:26376297

  15. Crystal structure of histidyl-tRNA synthetase from Escherichia coli complexed with histidyl-adenylate.

    PubMed Central

    Arnez, J G; Harris, D C; Mitschler, A; Rees, B; Francklyn, C S; Moras, D

    1995-01-01

    The crystal structure at 2.6 A of the histidyl-tRNA synthetase from Escherichia coli complexed with histidyl-adenylate has been determined. The enzyme is a homodimer with a molecular weight of 94 kDa and belongs to the class II of aminoacyl-tRNA synthetases (aaRS). The asymmetric unit is composed of two homodimers. Each monomer consists of two domains. The N-terminal catalytic core domain contains a six-stranded antiparallel beta-sheet sitting on two alpha-helices, which can be superposed with the catalytic domains of yeast AspRS, and GlyRS and SerRS from Thermus thermophilus with a root-mean-square difference on the C alpha atoms of 1.7-1.9 A. The active sites of all four monomers are occupied by histidyl-adenylate, which apparently forms during crystallization. The 100 residue C-terminal alpha/beta domain resembles half of a beta-barrel, and provides an independent domain oriented to contact the anticodon stem and part of the anticodon loop of tRNA(His). The modular domain organization of histidyl-tRNA synthetase reiterates a repeated theme in aaRS, and its structure should provide insight into the ability of certain aaRS to aminoacylate minihelices and other non-tRNA molecules. Images PMID:7556055

  16. Structure of the adenylation domain of NAD(+)-dependent DNA ligase from Staphylococcus aureus.

    PubMed

    Han, Seungil; Chang, Jeanne S; Griffor, Matt

    2009-11-01

    DNA ligase catalyzes phosphodiester-bond formation between immediately adjacent 5'-phosphate and 3'-hydroxyl groups in double-stranded DNA and plays a central role in many cellular and biochemical processes, including DNA replication, repair and recombination. Bacterial NAD(+)-dependent DNA ligases have been extensively characterized as potential antibacterial targets because of their essentiality and their structural distinction from human ATP-dependent DNA ligases. The high-resolution structure of the adenylation domain of Staphylococcus aureus NAD(+)-dependent DNA ligase establishes the conserved domain architecture with other bacterial adenylation domains. Two apo crystal structures revealed that the active site possesses the preformed NAD(+)-binding pocket and the 'C2 tunnel' lined with hydrophobic residues: Leu80, Phe224, Leu287, Phe295 and Trp302. The C2 tunnel is unique to bacterial DNA ligases and the Leu80 side chain at the mouth of the tunnel points inside the tunnel and forms a narrow funnel in the S. aureus DNA ligase structure. Taken together with other DNA ligase structures, the S. aureus DNA ligase structure provides a basis for a more integrated understanding of substrate recognition and catalysis and will be also be of help in the development of small-molecule inhibitors.

  17. Cloning, chromosomal mapping, and expression of human fetal brain type I adenylyl cyclase

    SciTech Connect

    Villacres, E.C.; Xia, Z.; Bookbinder, L.H.; Edelhoff, S.; Disteche, C.M.; Storm, D.R.

    1993-05-01

    The neural-specific calmodulin-sensitive adenylyl cyclase (type I), which was first cloned from bovine brain, has been implicated in learning and memory. The objective of this study was to clone and determine the chromosomal localization of human fetal brain type I adenylyl cyclase. A 3.8-kb cDNA clone was isolated that contained sequence coinciding with the 3{prime} end 2553 nucleotides of the bovine open reading frame. This clone shows 87% nucleotide and 92% translated amino acid sequence identity to the bovine clone. The most significant sequence differences were in the carboxy-terminal 100 amino acid residues. This region contains one of several possible calmodulin binding domains and the only putative cAMP-dependent protein kinase A phosphorylation site. A chimera was constructed that contained the 5{prime} half of the bovine type I adenylyl cyclase and the 3{prime} half of the human type I adenylyl cyclase. The activity of the chimeric gene product and its sensitivity to calmodulin and calcium were indistinguishable from those of the bovine type I adenylyl cyclase. In situ hybridization was used to localize the human type I adenylyl cyclase gene to the proximal portion of the short arm of chromosome 7. 36 refs., 4 figs.

  18. Production and Characterization of Monoclonal Antibodies to Soluble Rat Lung Guanylate Cyclase

    NASA Astrophysics Data System (ADS)

    Brandwein, Harvey; Lewicki, John; Murad, Ferid

    1981-07-01

    Four monoclonal antibodies to rat lung soluble guanylate cyclase [GTP pyrophosphate-lyase (cyclizing) EC 4.6.1.2] have been produced by fusing spleen cells from immunized BALB/c mice with SP-2/0 myeloma cells. The antibodies were detected by their ability to bind immobilized guanylate cyclase and by immunoprecipitation of purified enzyme in the presence of second (rabbit anti-mouse) antibody. After subcloning by limiting dilution, hybridomas were injected intraperitoneally into mice to produce ascitic fluid containing 2-5 mg of antibody per ml. The four antibodies obtained had titers of between 1:1580 and 1:3160 but were detectable at dilutions greater than 1:20,000. Soluble guanylate cyclase from several rat tissues were crossreactive with the four monoclonal antibodies, suggesting that the soluble enzyme from different rat tissues is antigenically similar. The antibodies also recognized soluble lung enzyme from rat, beef, and pig, while enzyme from rabbit was not crossreactive and mouse enzyme was recognized by only one of the antibodies. Particulate guanylate cyclase from a number of tissues had only minimal crossreactivity with the antibodies. Immunoprecipitated guanylate cyclase retained catalytic activity, could be activated with sodium nitroprusside, and was inhibited by cystamine. None of the antibodies were inhibitory under the conditions examined. These antibodies will be useful probes for the study of guanylate cyclase regulation and function under a variety of physiological conditions.

  19. Structural studies of intermediates along the cyclization pathway of Aplysia ADP-ribosyl cyclase.

    PubMed

    Kotaka, Masayo; Graeff, Richard; Chen, Zhe; Zhang, Li He; Lee, Hon Cheung; Hao, Quan

    2012-01-20

    Cyclic ADP-ribose (cADPR) is a calcium messenger that can mobilize intracellular Ca²⁺ stores and activate Ca²⁺ influx to regulate a wide range of physiological processes. Aplysia cyclase is the first member of the ADP-ribosyl cyclases identified to catalyze the cyclization of NAD⁺ into cADPR. The catalysis involves a two-step reaction, the elimination of the nicotinamide ring and the cyclization of the intermediate resulting in the covalent attachment of the purine ring to the terminal ribose. Aplysia cyclase exhibits a high degree of leniency towards the purine base of its substrate, and the cyclization reaction takes place at either the N1- or the N7-position of the purine ring. To decipher the mechanism of cyclization in Aplysia cyclase, we used a crystallization setup with multiple Aplysia cyclase molecules present in the asymmetric unit. With the use of natural substrates and analogs, not only were we able to capture multiple snapshots during enzyme catalysis resulting in either N1 or N7 linkage of the purine ring to the terminal ribose, we were also able to observe, for the first time, the cyclized products of both N1 and N7 cyclization bound in the active site of Aplysia cyclase.

  20. PKA and cAMP/CNG Channels Independently Regulate the Cholinergic Ca(2+)-Response of Drosophila Mushroom Body Neurons(1,2,3).

    PubMed

    Pavot, Pierre; Carbognin, Elena; Martin, Jean-René

    2015-01-01

    The mushroom bodies (MBs), one of the main structures in the adult insect brain, play a critical role in olfactory learning and memory. Though historical genes such as dunce and rutabaga, which regulate the level of cAMP, were identified more than 30 years ago, their in vivo effects on cellular and physiological mechanisms and particularly on the Ca(2+)-responses still remain largely unknown. In this work, performed in Drosophila, we took advantage of in vivo bioluminescence imaging, which allowed real-time monitoring of the entire MBs (both the calyx/cell-bodies and the lobes) simultaneously. We imaged neuronal Ca(2+)-activity continuously, over a long time period, and characterized the nicotine-evoked Ca(2+)-response. Using both genetics and pharmacological approaches to interfere with different components of the cAMP signaling pathway, we first show that the Ca(2+)-response is proportional to the levels of cAMP. Second, we reveal that an acute change in cAMP levels is sufficient to trigger a Ca(2+)-response. Third, genetic manipulation of protein kinase A (PKA), a direct effector of cAMP, suggests that cAMP also has PKA-independent effects through the cyclic nucleotide-gated Ca(2+)-channel (CNG). Finally, the disruption of calmodulin, one of the main regulators of the rutabaga adenylate cyclase (AC), yields different effects in the calyx/cell-bodies and in the lobes, suggesting a differential and regionalized regulation of AC. Our results provide insights into the complex Ca(2+)-response in the MBs, leading to the conclusion that cAMP modulates the Ca(2+)-responses through both PKA-dependent and -independent mechanisms, the latter through CNG-channels.

  1. PKA and cAMP/CNG Channels Independently Regulate the Cholinergic Ca2+-Response of Drosophila Mushroom Body Neurons1,2,3

    PubMed Central

    Pavot, Pierre; Carbognin, Elena

    2015-01-01

    Abstract The mushroom bodies (MBs), one of the main structures in the adult insect brain, play a critical role in olfactory learning and memory. Though historical genes such as dunce and rutabaga, which regulate the level of cAMP, were identified more than 30 years ago, their in vivo effects on cellular and physiological mechanisms and particularly on the Ca2+-responses still remain largely unknown. In this work, performed in Drosophila, we took advantage of in vivo bioluminescence imaging, which allowed real-time monitoring of the entire MBs (both the calyx/cell-bodies and the lobes) simultaneously. We imaged neuronal Ca2+-activity continuously, over a long time period, and characterized the nicotine-evoked Ca2+-response. Using both genetics and pharmacological approaches to interfere with different components of the cAMP signaling pathway, we first show that the Ca2+-response is proportional to the levels of cAMP. Second, we reveal that an acute change in cAMP levels is sufficient to trigger a Ca2+-response. Third, genetic manipulation of protein kinase A (PKA), a direct effector of cAMP, suggests that cAMP also has PKA-independent effects through the cyclic nucleotide-gated Ca2+-channel (CNG). Finally, the disruption of calmodulin, one of the main regulators of the rutabaga adenylate cyclase (AC), yields different effects in the calyx/cell-bodies and in the lobes, suggesting a differential and regionalized regulation of AC. Our results provide insights into the complex Ca2+-response in the MBs, leading to the conclusion that cAMP modulates the Ca2+-responses through both PKA-dependent and -independent mechanisms, the latter through CNG-channels. PMID:26464971

  2. Adenylyl cyclase-associated protein Aca1 regulates virulence and differentiation of Cryptococcus neoformans via the cyclic AMP-protein kinase A cascade.

    PubMed

    Bahn, Yong-Sun; Hicks, Julie K; Giles, Steven S; Cox, Gary M; Heitman, Joseph

    2004-12-01

    The evolutionarily conserved cyclic AMP (cAMP) signaling pathway controls cell functions in response to environmental cues in organisms as diverse as yeast and mammals. In the basidiomycetous human pathogenic fungus Cryptococcus neoformans, the cAMP pathway governs virulence and morphological differentiation. Here we identified and characterized adenylyl cyclase-associated protein, Aca1, which functions in parallel with the Galpha subunit Gpa1 to control the adenylyl cyclase (Cac1). Aca1 interacted with the C terminus of Cac1 in the yeast two-hybrid system. By molecular and genetic approaches, Aca1 was shown to play a critical role in mating by regulating cell fusion and filamentous growth in a cAMP-dependent manner. Aca1 also regulates melanin and capsule production via the Cac1-cAMP-protein kinase A pathway. Genetic epistasis studies support models in which Aca1 and Gpa1 are necessary and sufficient components that cooperate to activate adenylyl