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Sample records for adenylate cyclase activating

  1. Pituitary Adenylate Cyclase Activating Polypeptide

    PubMed Central

    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

    2010-01-01

    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. PMID:20889562

  2. 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

  3. Conformational basis for the activation of adenylate cyclase by adenosine

    PubMed Central

    Miles, D. L.; Miles, D. W.; Eyring, H.

    1977-01-01

    The ability of adenosine to stimulate adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] and increase adenosine 3′:5′-cyclic monophosphate (cAMP) levels has important biochemical consequences. These include the suppression of immune responses and cardiovascular effects. Recent investigations involving the ability of adenosine and adenosine analogs to stimulate adenylate cyclase provided experimental data that appear to be correlated with the ability of adenosine and analogs of adenosine to exist in the glycosidic high anti conformation. 9-β-D-Arabinofuranosyladenine, which is not stable in the high anti conformation, is inactive as a stimulator of adenylate cyclase. 2′-Deoxyadenosine is also not stable in the high anti conformation but its instability may be significantly decreased by intramolecular adjustments promoted by receptor or active site interactions. 2′-Deoxyadenosine does not activate adenylate cyclase in lymphocytes when ATP is the substrate but is able to activate adenylate cyclase when 2-fluoro ATP is the substrate. The inability of certain analogs of adenosine, with bulky groups substituted for hydrogen at the 8 position of the adenine base, to activate adenylate cyclase and increase either lymphocyte or cardiac cell cAMP levels is consistent with the designation of the high anti conformation as being the conformation required for the activation of adenylate cyclase. An understanding of the glycosidic conformation required by the extracellular adenosine receptor of the adenosine molecule provides the basis for designing nucleoside analogs of adenosine that will exert a desired effect on cAMP levels. The avoidance of unwanted immunosuppressive or cardiotoxic effects can be arranged by structural changes that prohibit the high anti conformation. PMID:267918

  4. 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.

  5. Plant adenylate cyclases.

    PubMed

    Lomovatskaya, Lidiya A; Romanenko, Anatoliy S; Filinova, Nadejda V

    2008-01-01

    Adenylate cyclase (AC) (ATP diphosphate-lyase cyclizing; EC 4.6.1.1) is a key component of the adenylate cyclase signaling system and catalyzes the generation of cyclic adenosine monophosphate (cAMP) from ATP. This review summarizes data from the literature and the authors' laboratory on the investigation of plant transmembrane (tmAC) and soluble (sAC) adenylate cyclases, in comparison with some key characteristics of adenylate cyclases of animal cells. Plant sAC has been demonstrated to exhibit similarities with animal sAC with respect to certain characteristics. External factors, such as far-red and red light, temperature, exogenous phytohormones, as well as specific triggering compounds of fungal and bacterial origin exert a significant influence on the activity of plant tmAC and sAC.

  6. Phospholipase A activity of adenylate cyclase toxin mediates translocation of its adenylate cyclase domain

    PubMed Central

    González-Bullón, David; Uribe, Kepa B.; Martín, César

    2017-01-01

    Adenylate cyclase toxin (ACT or CyaA) plays a crucial role in respiratory tract colonization and virulence of the whooping cough causative bacterium Bordetella pertussis. Secreted as soluble protein, it targets myeloid cells expressing the CD11b/CD18 integrin and on delivery of its N-terminal adenylate cyclase catalytic domain (AC domain) into the cytosol, generates uncontrolled toxic levels of cAMP that ablates bactericidal capacities of phagocytes. Our study deciphers the fundamentals of the heretofore poorly understood molecular mechanism by which the ACT enzyme domain directly crosses the host cell membrane. By combining molecular biology, biochemistry, and biophysics techniques, we discover that ACT has intrinsic phospholipase A (PLA) activity, and that such activity determines AC translocation. Moreover, we show that elimination of the ACT–PLA activity abrogates ACT toxicity in macrophages, particularly at toxin concentrations close to biological reality of bacterial infection. Our data support a molecular mechanism in which in situ generation of nonlamellar lysophospholipids by ACT–PLA activity into the cell membrane would form, likely in combination with membrane-interacting ACT segments, a proteolipidic toroidal pore through which AC domain transfer could directly take place. Regulation of ACT–PLA activity thus emerges as novel target for therapeutic control of the disease. PMID:28760979

  7. 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.

  8. 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.

  9. The influence of EDTA on adenylate cyclase activity in membranes from rat and mouse myocardium.

    PubMed

    Morris, S A; Bilezikian, J P

    1987-11-12

    Inclusion of EDTA in the homogenization buffer of both mouse and rat myocardium profoundly alters the properties of the adenylate cyclase complex. EDTA leads to an increase in the Vmax for adenylate cyclase activity due to all of the following agents: isoproterenol, Gpp[NH]p, forskolin and Mg2+. For forskolin and Mg2+, the EDTA-associated increase in Vmax is not accompanied by a change in sensitivity to activation. However, EDTA is associated with enhanced sensitivity to activation by isoproterenol and increased sensitivity to the effect of Mg2+ on isoproterenol-dependent adenylate cyclase activity. A result of greater isoproterenol-dependent adenylate cyclase activity, due to the presence of EDTA, is an attenuated synergistic contribution of Gpp[NH]p. Changes in stimulatable adenylate cyclase activity as a result of EDTA occurs in concert with effects of cholera toxin upon ADPribosylation of the guanine nucleotide regulatory protein, Ns. Substantial auto-ADP-ribosylation occurs in a cholera toxin-sensitive 42 kDa band in membranes prepared in the presence of EDTA. In addition, cofactor and substrate requirements in the cholera toxin-dependent ADP-ribosylation reaction depend on the method of membrane preparation. The results suggest that the integrity of the adenylate cyclase complex depends in part on the attention given to proteolysis that may be activated during the course of homogenization.

  10. 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

  11. Inhibition of forskolin-stimulated adenylate cyclase activity by 5-HT receptor agonists.

    PubMed

    Devivo, M; Maayani, S

    1985-12-17

    We measured the inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig hippocampal membranes by 5-HT, 5-carboxamidotryptamine (CAT) and 8-hydroxy-2-(di-n-propylamino) tetralin (PAT). Low concentrations of these agonists inhibited forskolin-stimulated adenylate cyclase activity in a concentration-dependent and saturable manner. The antagonist spiperone shifted the concentration-response curve to CAT to the right in a parallel manner. The EC50 values of CAT, PAT and 5-HT and the KB of spiperone suggest that this receptor may correspond to the 5-HT1A binding site.

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

    PubMed

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

    1978-06-01

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

  13. [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.

  14. 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.

  15. 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.

  16. 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.

  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. 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

  19. Enhanced serotonin-stimulated adenylate cyclase activity in membranes from adult guinea pig hippocampus.

    PubMed

    Shenker, A; Maayani, S; Weinstein, H; Green, J P

    1983-05-16

    We have developed an assay for serotonin (5-HT) stimulation of adenylate cyclase activity in membranes from adult guinea pig hippocampus. The response to 5-HT is concentration-dependent, with an EC50 of 0.01 microM, a shallow slope, and mean maximal stimulation of 90% over basal activity. The response to 5-HT is GTP-dependent and additive to the maximal stimulation by histamine. Micromolar concentrations of the known 5-HT receptor agonists, tryptamine and 5-methoxytryptamine, also stimulate cAMP production in this system, and their effect is not additive to that elicited by a maximal concentration of 5-HT. These results are consistent with the hypothesis that the response to 5-HT is elicited through a distinct receptor coupled to adenylate cyclase; the magnitude and the reproducibility of the 5-HT response in this system should make it useful for receptor classification. To examine the effect of prior exposure to endogenous 5-HT on the responsiveness of the system, we assayed 5-HT stimulation of enzyme activity in membranes prepared from animals 25-27 hrs after treatment with a single injection of reserpine (5 mg/kg, i.p.). The mean maximal stimulation of adenylate cyclase by 5-HT was increased to 150% over basal activity with no effect on the EC50 or slope of the 5-HT concentration-response curve. Reserpine pretreatment did not affect basal activity or histamine-stimulated adenylate cyclase activity. These results are discussed in the context of a hypothesis that endogenous 5-HT normally exerts a desensitizing effect on its receptors in situ.

  20. Inhibition of denuded mouse oocyte meiotic maturation by forskolin, an activator of adenylate cyclase.

    PubMed

    Urner, F; Herrmann, W L; Baulieu, E E; Schorderet-Slatkine, S

    1983-09-01

    Forskolin, a diterpene that activates rapidly adenylate cyclase activity in several somatic cell systems, prevents spontaneous meiotic maturation of denuded mouse oocytes (ED50 of inhibition approximately 2.5 microM), unlike cholera toxin. The oocyte is sensitive to the action of forskolin during the period preceding germinal vesicle breakdown (GVBD). Washing of the cells abolishes the effect. The diterpene potentiates the inhibitory effect of iso-butyl-methyl-xanthine (IBMX), a phosphodiesterase inhibitor, and it increases cAMP concentration in the oocytes. These findings not only confirm the antagonistic effect of cAMP on the first step of meiosis reinitiation (GVBD) in mammalian oocytes, but also provide the first demonstration of a functional adenylate cyclase system in mammalian oocytes upon which regulatory signals may act.

  1. Molecular cloning of an orphan G-protein-coupled receptor that constitutively activates adenylate cyclase.

    PubMed Central

    Eggerickx, D; Denef, J F; Labbe, O; Hayashi, Y; Refetoff, S; Vassart, G; Parmentier, M; Libert, F

    1995-01-01

    A human gene encoding an orphan G-protein-coupled receptor named ACCA (adenylate cyclase constitutive activator) was isolated from a genomic library using as a probe a DNA fragment obtained by low-stringency PCR. Human ACCA (hACCA) is a protein of 330 amino acids that exhibits all the structural hallmarks of the main family of G-protein-coupled receptors. Expression of hACCA resulted in a dramatic stimulation of adenylate cyclase, similar in amplitude to that obtained with other Gs-coupled receptors fully activated by their respective ligands. This stimulation was obtained in a large variety of stable cell lines derived from various organs, and originating from different mammalian species. hACCA was found to be the human homologue of a recently reported mouse orphan receptor (GPCR21). The mouse ACCA (mACCA) was therefore recloned by PCR, and expression of mACCA in Cos-7 cells demonstrated that the mouse receptor behaved similarly as a constitutive activator of adenylate cyclase. It is not known presently whether the stimulation of adenylate cyclase is the result of a true constitutive activity of the receptor or, alternatively, is the consequence of a permanent stimulation by a ubiquitous ligand. The tissue distribution of mACCA was determined by RNase protection assay. Abundant transcripts were found in the brain, whereas lower amounts were detected in testis, ovary and eye. Various hypotheses concerning the constitutive activity of ACCA and their potential biological significance are discussed. Images Figure 4 Figure 5 PMID:7639700

  2. Antagonism of histamine-activated adenylate cyclase in brain by D-lysergic acid diethylamide.

    PubMed

    Green, J P; Johnson, C L; Weinstein, H; Maayani, S

    1977-12-01

    D-Lysergic acid diethylamide and D-2-bromolysergic acid diethylamide are competitive antagonists of the histamine activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); E.C. 4.6.1.1] in broken cell preparations of the hippocampus and cortex of guinea pig brain. The adenylate cyclase is linked to the histamine H2-receptor. Both D-lysergic acid diethylamide and D-2-bromolysergic acid diethylamide show topological congruency with potent H2-antagonists. D-2-Bromolysergic acid diethylamide is 10 times more potent as an H2-antagonist than cimetidine, which has been the most potent H2-antagonist reported, and D-lysergic acid diethylamide is about equipotent to cimetidine. Blockade of H2-receptors could contribute to the behavioral effects of D-2-bromolysergic acid diethylamide and D-lysergic acid diethylamide.

  3. Antagonism of histamine-activated adenylate cyclase in brain by D-lysergic acid diethylamide.

    PubMed Central

    Green, J P; Johnson, C L; Weinstein, H; Maayani, S

    1977-01-01

    D-Lysergic acid diethylamide and D-2-bromolysergic acid diethylamide are competitive antagonists of the histamine activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); E.C. 4.6.1.1] in broken cell preparations of the hippocampus and cortex of guinea pig brain. The adenylate cyclase is linked to the histamine H2-receptor. Both D-lysergic acid diethylamide and D-2-bromolysergic acid diethylamide show topological congruency with potent H2-antagonists. D-2-Bromolysergic acid diethylamide is 10 times more potent as an H2-antagonist than cimetidine, which has been the most potent H2-antagonist reported, and D-lysergic acid diethylamide is about equipotent to cimetidine. Blockade of H2-receptors could contribute to the behavioral effects of D-2-bromolysergic acid diethylamide and D-lysergic acid diethylamide. Images PMID:23536

  4. 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.

  5. 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

  6. 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

  7. Adenylate cyclase activity along the rabbit nephron as measured in single isolated segments.

    PubMed

    Imbert, M; Chabardès, D; Montégut, M; Clique, A; Morel, F

    1975-01-01

    A method is described, which allows adenylate cyclase activity measurement in single pieces of various nephron segments. Tubular samples of 0.5 to 2 mm length were isolated by microdissection from collagenase treated slices of rabbit kidney. A photograph of each piece was taken in order to measure its length. After a permeabilisation treatment involving preincubation in a hypoosmotic medium and a freezing step, each sample was incubated for 30 mm at 30 degrees C in a medium containing high specific (alpha-32-P)-ATP 3-10-4 M, final volume 2.5 mu 1. The (32P)-cAMP formed was separated from the other labelled nucleotides by filtering the incubate on a dry aluminium oxide microcolumn, 3H cAMP was added as a tracer for measuring cAMP recovery. The sensitivity of the method was found to be a few fentomoles (10-15 M) cAMP. cAMP generation increased linearly as a function of the incubation time up to more than 30 min, and as a function of the length of the segment used. Control and fluoride (5 mM) stimulated adenvlate cyclase activities were measured in the following segments of the nephron: early proximal convoluted tubule (PCT), pars recta of the proximal tubule (PR), thin descending limb of the loop (TDL), cortical portion of the thick ascending limb (CAL), distal convoluted tubule (dct), first branched portion of the collecting tubule (BCT), further cortical (CCT) and medullary (MCT) portions of the collecting tubule. Mean control adenylate cyclase activity varied from 7 (PR) to 75 (BCT) fmoles/mm/30 min. Flouride addition resulted in a 10 (BCT) to 50 (PR) fold increase in enzyme activity. Series of replicates gave a scatter equal to plus or minus 20% (S.D. as a per cent of the mean). The method described appears to be suitable to determine which nephron segments contain hormone-dependent adenylate cyclase.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. Ontogenic development of antidiuretic hormone receptors in rat kidney: comparison of hormonal binding and adenylate cyclase activation.

    PubMed

    Rajerison, R M; Butlen, D; Jard, S

    1976-03-01

    The development of adenylate cyclase responsiveness to vasopressin and parathyroid hormone was studied using membrane fractions prepared from medullo-papillary and cortical portions of kidneys of 2-46-day-old rats. The development of vasopressin binding capacity was followed on the same preparations, using [3H]vasopressin. The characteristics of medullo-papillary adenylate cyclase response to vasopressin were identical in young and adult control animals as regards apparent Km values for [Lys8]vasopressin (3 X 10(-8) M), specificity towards the natural neurohypophysial peptides and the effects of Mg2+. However, the magnitude of maximal enzyme activation by vasopressin was much lower in very young than adult animals. Accordingly vasopressin responsiveness increased sharply between the 10th and 25th days but the magnitude of the maximal response only reached the adult value between the 30th and 45th days after birth. During both periods basal adenylate cyclase activity was almost independent of age. Specific vasopressin binding sites were detected on kidney medullo-papillary membranes from young animals. Vasopressin binding capacity and adenylate cyclase responsiveness to the hormone followed similar development patterns. However, the appearance of specific binding sites slightly preceded the onset of adenylate cyclase responsiveness. Basal cortical adenylate cyclase activity/mg protein was 12 times higher in 2-day-old rats than in the adult controls. It dropped with age but only fell to the adult value between the 25th and the 35th days after birth. For the youngest animals tested (2 days old), the increase in activity due to parathyroid hormone was about half the increase measured in adults, and gradually rose to about 75% of the adult response between the 2nd and 46th days after birth. Apparent Km values for parathyroid hormone were identical in young and adult animals (3.2 and 3.0 U/ml, respectively).

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Cloning and characterization of a Drosophila serotonin receptor that activates adenylate cyclase.

    PubMed Central

    Witz, P; Amlaiky, N; Plassat, J L; Maroteaux, L; Borrelli, E; Hen, R

    1990-01-01

    Using a strategy based on nucleotide sequence homology between genes encoding receptors that interact with guanine nucleotide-binding proteins, we have isolated Drosophila genomic and cDNA clones encoding a functional serotonin receptor (5HT-dro receptor). This protein is expressed predominantly in Drosophila heads and exhibits highest homology with the human 5HT1A receptor. The predicted structure of the 5HT-dro receptor reveals two unusual features: (i) eight putative transmembrane domains instead of the expected seven and (ii) a Gly-Ser repeat that is a potential glycosaminoglycan attachment site. When stably introduced into mouse NIH 3T3 cells, the 5HT-dro receptor activates adenylate cyclase in response to serotonin and is inhibited by serotonin receptor antagonists such as dihydroergocryptine. The 5HT-dro receptor or closely related receptors might be responsible for the serotonin-sensitive cyclase that has been suggested to play a role in learning and modulation of circadian rhythm in a number of invertebrate systems. Images PMID:2174167

  1. Adenylate cyclase in striatal cholinergic interneurons regulates acetylcholine release.

    PubMed

    Login, I S; Hewlett, E L

    1996-10-07

    Fractional [3H]ACH efflux from dissociated rat striata tested whether tonic inhibition prevents stimulation of acetylcholine (ACH) release by adenylate cyclase. Forskolin stimulated release from the dissociated cells (threshold at 300 nM; EC50 > or = 1 MicroM). Release was also stimulated by 3-isobutyl-1-methylxanthine and was additive with forskolin. The 1,9-dideoxy forskolin analog that lacks cyclase-stimulating activity was ineffective. Thus, stimulation of adenylate cyclase within striatal cholinergic interneurons increases ACH secretion but is tonically inhibited by endogenous striatal transmitters. Disinhibition of the excitatory cyclase by denervation of striatal cholinergic interneurons in situ could contribute to supersensitivity without receptor upregulation.

  2. Cytochemical localization of adenylate cyclase activity in heart tissue with cerium.

    PubMed

    Schulze, W; Will-Shahab, L; Küttner, I

    1986-01-01

    Adenylate cyclase (AC) activity showed a doses depending inactivation of the basal activity and of the sodium fluoride stimulation by cerium in homogenates of unfixed and fixed guinea pig hearts. The isoproterenol and guanine nucleotide stimulation was not more than two times of the basal activity in glutaraldehyde-prefixed heart homogenates in the presence of 2 mmol/l CeCl3. The inactivation of the AC (activity) by cerium was less than in the presence of lead. Test tube experiments showed no differences in the precipitation of imidodiphosphate in comparison with inorganic phosphate. The substrate AMP-PNP was not spontaneously hydrolysed by 2 mmol/l CeCl3. Ultrastructural analysis of cytochemical incubation of glutaraldehyde-fixed slices and small pieces of guinea pig heart tissue showed fine-amorphous precipitations of reaction products localized along the plasma membrane of the sarcolemma, the nexuses of the intercalated discs and the T-tubule membranes. No precipitates were found neither on the junctional nor on other SR membranes. Nonspecific coarse and clumped precipitates have been detected in the intercellular space on components of the basal membranes. It was not able to demonstrate cytochemically stimulation of AC by hormones or by sodium fluoride. The localization of the basal AC activity in heart tissue seems to be better with cerium as capture agent than with lead. However, differences in the localization of the AC activity in heart tissue were not observed.

  3. 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

  4. 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. PMID:28400813

  5. Cellular localization of pituitary adenylate cyclase-activating peptide (PACAP) following traumatic brain injury in humans.

    PubMed

    van Landeghem, Frank K H; Weiss, Thorsten; Oehmichen, Manfred; von Deimling, Andreas

    2007-06-01

    The pituitary adenylate cyclase-activating peptide (PACAP) is involved in many processes of the developing and mature central nervous system, such as proliferation, differentiation, apoptosis, neurotransmission, inflammation and neuroprotection. Alternative posttranslational processing of PACAP results in two biologically active, amidated 27- and 38-amino acid peptides termed PACAP27 and PACAP38. In the present study, we examined whether traumatic brain injury (TBI) affects cellular immunopositivity for PACAP27 and PACAP38. Patients (n = 55) were classified into three groups dependent on their survival time (under 24 h, between 24 h and 7 days and between 7 days and 99 days postinjury). PACAP27 and PACAP38 were expressed by neurons and glial cells in normal human neocortex (n = 10). Following TBI, the total number of PACAP27- and PACAP38-positive cells was significantly decreased for a prolonged survival period within the traumatized neocortex. In the pericontusional cortex, the number of cells expressing PACAP27 and PACAP38 was significantly increased at all survival times examined. Triple immunofluorescence examinations revealed a significant increase in the absolute numbers of GFAP-positive reactive astrocytes as well as a decrease in the CNP-positive oligodendrocytes, each coexpressing PACAP27 or PACAP38 in the contusional and pericontusional cortex. We hypothesize that the increase of glial PACAP immunoreactivity may be interpreted as part of a complex endogenous neuroprotective response in the pericontusional regions, but the precise role of PACAP following TBI is yet to be determined.

  6. 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.

  7. 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.

  8. Posttraumatic administration of pituitary adenylate cyclase activating polypeptide in central fluid percussion injury in rats.

    PubMed

    Kövesdi, Erzsébet; Tamás, Andrea; Reglodi, Dóra; Farkas, Orsolya; Pál, József; Tóth, Gábor; Bukovics, Péter; Dóczi, Tamás; Büki, András

    2008-04-01

    Several in vitro and in vivo experiments have demonstrated the neuroprotective effects of pituitary adenylate cyclase activating polypeptide (PACAP) in focal cerebral ischemia, Parkinson's disease and traumatic brain injury (TBI). The aim of the present study was to analyze the effect of PACAP administration on diffuse axonal injury (DAI), an important contributor to morbidity and mortality associated with TBI, in a central fluid percussion (CFP) model of TBI. Rats were subjected to moderate (2 Atm) CFP injury. Thirty min after injury, 100 microg PACAP was administered intracerebroventricularly. DAI was assessed by immunohistochemical detection of beta-amyloid precursor protein, indicating impaired axoplasmic transport, and RMO-14 antibody, representing foci of cytoskeletal alterations (neurofilament compaction), both considered classical markers of axonal damage. Analysis of damaged, immunoreactive axonal profiles revealed significant axonal protection in the PACAP-treated versus vehicle-treated animals in the corticospinal tract, as far as traumatically induced disturbance of axoplasmic transport and cytoskeletal alteration were considered. Similarly to our former observations in an impact acceleration model of diffuse TBI, the present study demonstrated that PACAP also inhibits DAI in the CFP injury model. The finding indicates that PACAP and derivates can be considered potential candidates for further experimental studies, or purportedly for clinical trials in the therapy of TBI.

  9. Pituitary adenylate cyclase-activating polypeptide is reduced in Alzheimer disease.

    PubMed

    Han, Pengcheng; Liang, Winnie; Baxter, Leslie C; Yin, Junxiang; Tang, Zhiwei; Beach, Thomas G; Caselli, Richard J; Reiman, Eric M; Shi, Jiong

    2014-05-13

    There is growing evidence that pituitary adenylate cyclase-activating polypeptide (PACAP) is associated with Alzheimer disease (AD) pathology in animal models, but human studies are needed. We studied the brains of patients with pathologically confirmed late-onset AD and age-matched cognitively normal (CN) subjects to investigate the expression of PACAP messenger RNA (34 AD and 14 CN) and protein (12 AD and 11 CN) in a case-control study. We report that PACAP levels are reduced in multiple brain regions, including the entorhinal cortex, the middle temporal gyrus, the superior frontal gyrus, and the primary visual cortex. This reduction is correlated with higher amyloid burden (CERAD plaque density) in the entorhinal cortex and superior frontal gyrus but not in the primary visual cortex, a region spared in most cases of AD. PACAP expression is lower in advanced Braak stages (V and VI) than in moderate stages (III and IV). Increased PACAP levels are associated with decreased scores on the Dementia Rating Scale, a global cognitive measure. Finally, CSF levels paralleled brain levels in AD but not in Parkinson dementia or frontotemporal dementia brains. The close relationship between PACAP reduction and the severity of AD pathology suggests that downregulation of PACAP may contribute to AD pathogenesis.

  10. Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Is Involved in Adult Mouse Hippocampal Neurogenesis After Stroke.

    PubMed

    Matsumoto, Minako; Nakamachi, Tomoya; Watanabe, Jun; Sugiyama, Koichi; Ohtaki, Hirokazu; Murai, Norimitsu; Sasaki, Shun; Xu, Zhifang; Hashimoto, Hitoshi; Seki, Tamotsu; Miyazaki, Akira; Shioda, Seiji

    2016-06-01

    In the subgranular zone (SGZ) of the hippocampus, neurogenesis persists throughout life and is upregulated following ischemia. Accumulating evidence suggests that enhanced neurogenesis stimulated by ischemic injury contributes to recovery after stroke. However, the mechanisms underlying the upregulation of neurogenesis are unclear. We have demonstrated that a neuropeptide, pituitary adenylate cyclase-activating polypeptide (PACAP), exerts a wide range of effects on neural stem cells (NSCs) during neural development. Here, we examined the effects of endogenous and exogenous PACAP in adult NSCs of the SGZ. Immunostaining showed expression of the PACAP receptor PAC1R in nestin-positive NSCs of adult naive mice. PACAP injection into the lateral ventricle increased bromodeoxyuridine (BrdU)-positive proliferative cells in the SGZ. These data suggest that PACAP promoted the proliferation of NSCs. In global ischemia model mice, the number of BrdU-positive cells was increased in wild-type mice but not in PACAP heterozygous knockout mice. The BrdU-positive cells that increased in number after ischemia were immunopositive for SOX2, a marker of NSCs, and differentiated into NeuN-positive mature neurons at 4 weeks after ischemia. These findings suggest that PACAP contributes to the proliferation of NSCs and may be associated with recovery after brain injury.

  11. Effect of hypolipidemic drugs on basal and stimulated adenylate cyclase activity in tumor cells

    SciTech Connect

    Bershtein, L.M.; Kovaleva, I.G.; Rozenberg, O.A.

    1986-02-01

    This paper studies adenylate cyclase acticvity in Ehrlich's ascites carcinoma (EAC) cells during administration of drugs with a hypolipidemic action. Seven to eight days before they were killed, male mice ingested the antidiabetic biguanide phenformin, and the phospholipid-containing preparation Essentiale in drinking water. The cAMP formed was isolated by chromatography on Silufol plates after incubation of the enzyme preparation with tritium-ATP, or was determined by the competitive binding method with protein. It is shown that despite the possible differences in the concrete mechanism of action of the hypolipidemic agents chosen for study on the cyclase system, the use of such agents, offers definite prospects for oriented modification of the hormone sensitivity of tumor cells.

  12. Comprehensive behavioral analysis of pituitary adenylate cyclase-activating polypeptide (PACAP) knockout mice.

    PubMed

    Hattori, Satoko; Takao, Keizo; Tanda, Koichi; Toyama, Keiko; Shintani, Norihito; Baba, Akemichi; Hashimoto, Hitoshi; Miyakawa, Tsuyoshi

    2012-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. PACAP is widely expressed throughout the brain and exerts its functions through the PACAP-specific receptor (PAC(1)). Recent studies reveal that genetic variants of the PACAP and PAC(1) genes are associated with mental disorders, and several behavioral abnormalities of PACAP knockout (KO) mice are reported. However, an insufficient number of backcrosses was made using PACAP KO mice on the C57BL/6J background due to their postnatal mortality. To elucidate the effects of PACAP on neuropsychiatric function, the PACAP gene was knocked out in F1 hybrid mice (C57BL/6J × 129SvEv) for appropriate control of the genetic background. The PACAP KO mice were then subjected to a behavioral test battery. PACAP deficiency had no significant effects on neurological screen. As shown previously, the mice exhibited significantly increased locomotor activity in a novel environment and abnormal anxiety-like behavior, while no obvious differences between genotypes were shown in home cage (HC) activity. In contrast to previous reports, the PACAP KO mice showed normal prepulse inhibition (PPI) and slightly decreased depression-like behavior. Previous study demonstrates that the social interaction (SI) in a resident-intruder test was decreased in PACAP KO mice. On the other hand, we showed that PACAP KO mice exhibited increased SI in Crawley's three-chamber social approach test, although PACAP KO had no significant impact on SI in a HC. PACAP KO mice also exhibited mild performance deficit in working memory in an eight-arm radial maze (RM) and the T-maze (TM), while they did not show any significant abnormalities in the left-right discrimination task in the TM. These results suggest that PACAP has an important role in the regulation of locomotor activity, social behavior, anxiety-like behavior and, potentially, working memory.

  13. Comprehensive behavioral analysis of pituitary adenylate cyclase-activating polypeptide (PACAP) knockout mice

    PubMed Central

    Hattori, Satoko; Takao, Keizo; Tanda, Koichi; Toyama, Keiko; Shintani, Norihito; Baba, Akemichi; Hashimoto, Hitoshi; Miyakawa, Tsuyoshi

    2012-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide acting as a neurotransmitter, neuromodulator, or neurotrophic factor. PACAP is widely expressed throughout the brain and exerts its functions through the PACAP-specific receptor (PAC1). Recent studies reveal that genetic variants of the PACAP and PAC1 genes are associated with mental disorders, and several behavioral abnormalities of PACAP knockout (KO) mice are reported. However, an insufficient number of backcrosses was made using PACAP KO mice on the C57BL/6J background due to their postnatal mortality. To elucidate the effects of PACAP on neuropsychiatric function, the PACAP gene was knocked out in F1 hybrid mice (C57BL/6J × 129SvEv) for appropriate control of the genetic background. The PACAP KO mice were then subjected to a behavioral test battery. PACAP deficiency had no significant effects on neurological screen. As shown previously, the mice exhibited significantly increased locomotor activity in a novel environment and abnormal anxiety-like behavior, while no obvious differences between genotypes were shown in home cage (HC) activity. In contrast to previous reports, the PACAP KO mice showed normal prepulse inhibition (PPI) and slightly decreased depression-like behavior. Previous study demonstrates that the social interaction (SI) in a resident-intruder test was decreased in PACAP KO mice. On the other hand, we showed that PACAP KO mice exhibited increased SI in Crawley's three-chamber social approach test, although PACAP KO had no significant impact on SI in a HC. PACAP KO mice also exhibited mild performance deficit in working memory in an eight-arm radial maze (RM) and the T-maze (TM), while they did not show any significant abnormalities in the left-right discrimination task in the TM. These results suggest that PACAP has an important role in the regulation of locomotor activity, social behavior, anxiety-like behavior and, potentially, working memory. PMID:23060763

  14. 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.

  15. Molecular cloning and expression of a chicken pituitary adenylate cyclase-activating polypeptide receptor.

    PubMed

    Peeters, K; Gerets, H H; Princen, K; Vandesande, F

    1999-08-25

    Although, since the isolation of pituitary adenylate cyclase-activating polypeptide (PACAP), a wealth of literature has been published describing its localization, binding sites, and biological activities in a variety of mammalian tissues, only very little is known about PACAP in avian species. Therefore, in order to find out the sites of actions of PACAP and to elucidate its physiological significance in birds, we identified a chicken PACAP receptor homologue of the mammalian type I receptors (PAC(1)-Rs). The chicken PACAP type I cDNA sequence was obtained using reverse transcriptase-polymerase chain reaction (RT-PCR) in combination with 3'- and 5'-RACE PCR. This cDNA encodes a 471 amino acid precursor protein, sharing 81-83% sequence identity with mammalian analogs and 76% amino acid identity with the goldfish type I PACAP receptor. Northern blot analysis of chicken brain poly(A)(+)-rich RNA revealed the presence of a 5.5 kb and 7.5 kb PAC(1) receptor transcript. RT-PCR revealed that the chicken PACAP receptor is mainly expressed in the brain and gonads. A smaller amount of the receptor mRNA was found in pituitary, adrenal gland, kidney, intestine, pancreas, lung, and heart tissue. In situ hybridization with specific antisense oligodeoxynucleotide probes showed a widespread distribution of PAC(1) receptor mRNA in the chicken brain, with the highest expression being found in the dorsal telencephalon, olfactory bulb, hypothalamus, optic tectum, and cerebellar cortex. These findings suggest that PACAP affect a variety of functions both in the brain and peripheral tissues of the chicken.

  16. 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.

  17. 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

  18. 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

  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. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. Adenylate cyclases involvement in pathogenicity, a minireview.

    PubMed

    Costache, Adriana; Bucurenci, Nadia; Onu, Adrian

    2013-01-01

    Cyclic AMP (cAMP), one of the most important secondary messengers, is produced by adenylate cyclase (AC) from adenosine triphosphate (ATP). AC is a widespread enzyme, being present both in prokaryotes and eukaryotes. Although they have the same enzymatic activity (ATP cyclization), the structure of these proteins varies, depending on their function and the producing organism. Some pathogenic bacteria utilize these enzymes as toxins which interact with calmodulin (or another eukaryote activator), causing intense cAMP synthesis and disruption of infected cell functions. In contrast, other pathogenic bacteria benefit of augmentation of AC activity for their own function. Based on sequence analysis ofAC catalytic domain from two pathogenic bacteria (Bacillus anthracis and Bordetellapertussis) with known three-dimensional structures, a possible secondary structure for 1-255 amino acid fragment from Pseudomonas aeruginosa AC (with 80TKGFSVKGKSS90 as the ATP binding site) is proposed.

  6. 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.

  7. 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

  8. Inhibition of human platelet adenylate cyclase activity by adrenaline, thrombin and collagen: analysis and reinterpretation of experimental data.

    PubMed Central

    Juska, A; Farndale, R W

    1999-01-01

    Mathematical models based on the current understanding of stimulation and inhibition of adenylate cyclase (AC) activity have been developed and used to analyse experimental data [Farndale, Winkler, Martin and Barnes (1992) Biochem. J. 282, 2532] describing the inhibition of human platelet AC by collagen, thrombin and adrenaline. Here it has been demonstrated that neither affinities of receptors specific for adrenaline or thrombin nor the activity of cAMP phosphodiesterase are affected by collagen. Both collagen and thrombin at high doses act as effective inhibitors of AC activity. Inhibition of AC activity by collagen proceeds via two parallel pathways; the same is true for thrombin at moderate concentrations, and the two ligands act independently. The G-protein-dependence of these pathways is distinct from that mediating inhibition of AC activity by adrenaline, i.e. Gi2. Convergence of the inhibitory pathways takes place at the catalytic subunit of AC. PMID:10391837

  9. [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.

  10. 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.

  11. 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

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

    PubMed

    Hammack, Sayamwong E; May, Victor

    2015-08-01

    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 in mediating the consequences of stressor exposure and relatedly, describe more recent studies suggesting that PACAP in the central nucleus of the amygdala may impact the emotional aspects of chronic pain states. In aggregate, these results are consistent with data suggesting that PACAP dysregulation is associated with posttraumatic stress disorder in humans. Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  13. Effects of cimetidine on adenylate cyclase activity of guinea pig gastric mucosa stimulated by histamine, sodium fluoride and 5'-guanylylimidodiphosphate.

    PubMed

    Anttila, P; Westermann, E

    1976-08-01

    Cimetidine, a recently developed histamine H2-receptor blocking agent has been shown to be a potent inhibitor of histamine-stimulated gastric acid secretion in rat, cat, dog and man. To study the mode of action of cimetidine the modification of stimulatory effects of histamine, sodium flouride and 5'-guanylylimidodiphosphate by cimetidine on the adenylate cyclase activity of guinea pig gastric mucosa was studied. The effect of cimetidine was also compared to that of metiamide, an older histamine H2-receptor antagonist. The effect of cimetidine was qualitatively similar to that of metiamide, i.e. a selective blockade of histamine H2-receptors. Quantitatively cimetidine was about 10-fold more potent than metiamide.

  14. 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

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

    PubMed

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

    2013-06-01

    Sulforaphane is a naturally occurring isothiocyanate, which can be found in cruciferous vegetables such as broccoli and cabbage. Sulforaphane was found to have very potent inhibitory effects on tumor growth through regulation of diverse mechanisms. However, no data are available concerning the effects of sulforaphane on platelet activation and its relative issues. Activation of platelets caused by arterial thrombosis is relevant to a variety of cardiovascular diseases. Hence, the aim of this study was to examine the in vivo antithrombotic effects of sulforaphane and its possible mechanisms in platelet activation. Sulforaphane (0.125 and 0.25 mg/kg) was effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice. Other in vivo studies also revealed that sulforaphane (0.25 mg/kg) significantly prolonged platelet plug formation in mice. In addition, sulforaphane (15-75 μM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen. Sulforaphane inhibited platelet activation accompanied by inhibiting relative Ca(2+) mobilization; phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs) and Akt; and hydroxyl radical (OH(●)) formation. Sulforaphane markedly increased cyclic (c)AMP, but not cyclic (c)GMP levels, and stimulated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, but not ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxal in-1-one), an inhibitor of guanylate cyclase, obviously reversed the sulforaphane-mediated effects on platelet aggregation; PKC activation, p38 MAPK, Akt and VASP phosphorylation; and OH(●) formation. Furthermore, a PI3-kinase inhibitor (LY294002) and a p38 MAPK inhibitor (SB203580) both significantly diminished PKC activation and p38 MAPK and Akt phosphorylation; in contrast, a PKC inhibitor (RO318220) did not diminish p38 MAPK or Akt phosphorylation stimulated by collagen. This

  16. Adenyl cyclase in the human placenta.

    PubMed

    Sato, K; Ryan, K J

    1971-09-21

    This study demonstrated that the human placenta possesses an adenyl cyclase system responsive to catecholamines and sodium flouride (NaF). 2.5 gm human term placentas were homogenized, centrifuged, washed, resuspended, and used as the enzyme system when placed with various agents. Incubations and the determination of adenosine 3', 5' monophosphate (cyclic AMP) formed were performed. Samples stimulated by .0001 M catecholamines (L-epinephrine or L-norepinephrine) or .01 M NaF had higher levels of cyclic AMP than the controls (p. 005 for catecholamine-treated samples and p. 001 for NaF-treated samples). A concentration of .0001 M L-epinephrine or L-norepinephrine appeared to be a maximum effective dose and .0000001 M a minimum. L=epinephrine was 10 times as effective in the stimulation as L-norepinephrine. With .0001 M, 499 and 439 pmoles/10 minutes per 25 mg of tissue was formed, whereas in the control (no added hormones) 256 pmoles/10 minutes were formed. 3.2% ethanol activated the system by a small amount (p.02). Propranolol alone did not appear to have any effect; however, the effect of .0001 M L-epinephrine was reduced by 95% in the presence of .00001 M propranolol. Propranolol had no effect on NaF-stimulated activity.

  17. 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.

  18. 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.

  19. Food restriction modulates. beta. -adrenergic-sensitive adenylate cyclase in rat liver during aging

    SciTech Connect

    Katz, M.S. Audie L. Murphy Memorial Veterans Hospital, San Antonio, TX )

    1988-01-01

    Adenylate cyclase activities were studied in rat liver during postmaturational aging of male Fischer 344 rats fed ad libitum or restricted to 60% of the ad libitum intake. Catecholamine-stimulated adenylate cyclase activity increased by 200-300% between 6 and 24-27 mo of age in ad libitum-fed rats, whereas in food-restricted rats catecholamine response increased by only 58-84% between 6 and 30 mo. In ad libitum-fed rats, glucagon-stimulated enzyme activity also increased by 40% between 6 and 12 mo and in restricted rats a similar age-related increase was delayed until 18 mo. {beta}-Adrenergic receptor density increased by 50% between 6 and 24 mo in livers from ad libitum-fed but not food-restricted rats and showed a highly significant correlation with maximal isoproterenol-stimulated adenylate cyclase activity over the postmaturational life span. Age-related increases in unstimulated (basal) adenylate cyclase activity and nonreceptor-mediated enzyme activation were retarded by food restriction. The results demonstrate that food restriction diminishes a marked age-related increase in {beta}-adrenergic-sensitive adenylate cyclase activity of rat liver. Alterations of adrenergic-responsive adenylate cyclase with age and the modulatory effects of food restriction appear to be mediated by changes in both receptor and nonreceptor components of adenylate cyclase.

  20. Characterization of the 5-hydroxytryptamine1a receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig and rat hippocampal membranes.

    PubMed

    De Vivo, M; Maayani, S

    1986-07-01

    The inhibition of forskolin-stimulated adenylate cyclase activity by 5-hydroxytryptamine (5-HT) receptor agonists was measured in guinea pig and rat hippocampal membranes. The results were consistent with the inhibition being mediated by a single, homogeneous population of receptors. In guinea pig hippocampal membranes 8-hydroxy-2-(di-n-propylamino)tetralin, d-lysergic acid diethylamide, 5-HT and buspirone were potent in inhibiting forskolin-stimulated adenylate cyclase activity, with EC50 values of 18, 24, 53 and 146 nM, respectively. Spiperone (Kb = 26 nM) and methiothepin (Kb = 13 nM) were potent competitive antagonists at this receptor whereas ketanserin, a high affinity 5-HT2 receptor ligand, and ICS 205-930, a high affinity peripheral neuronal (M) receptor ligand, were not. In rat hippocampal membranes, 8-hydroxy-2-(di-n-propylamino)tetralin, d-lysergic acid diethylamide, 5-HT and buspirone were potent agonists and exhibited the same rank order of potency as in guinea pig hippocampal membranes. The maximal percentage of inhibition by buspirone was significantly less than the maximal percentage of inhibition by 5-HT in rat membranes, suggesting that it is a partial agonist at this receptor, with an intrinsic activity relative to 5-HT of 0.5. The concentration-response data show that the inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig and rat hippocampal membranes is mediated by a receptor with the characteristics of the 5-HT1A binding site. We propose that the inhibition of adenylate cyclase activity is a functional correlate of this binding site. This response is suitable for measuring activities and affinities of drugs acting at 5-HT1A receptors.

  1. 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

  2. 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.

  3. Behavioral effects of local microinfusion of pituitary adenylate cyclase activating polypeptide (PACAP) into the paraventricular nucleus of the hypothalamus (PVN).

    PubMed

    Norrholm, Seth D; Das, Mahasweta; Légrádi, Gábor

    2005-05-15

    Pituitary adenylate cyclase activating polypeptide (PACAP) has been implicated in the regulation of several autonomic and neuroendocrine functions. In the hypothalamic paraventricular nucleus (PVN), for example, PACAP-immunoreactive fibers densely innervate corticotropin-releasing hormone (CRH)-containing neurons in the medial parvocellular region, suggesting that PACAP acts to mediate stress responses. Therefore, we examined the behavioral effects of an intra-PVN PACAP injection (25 pmol) in combination with a mild stressor. PACAP or artificial cerebrospinal fluid (aCSF) was microinjected into the PVN (0.25 l) and then animals were restrained or placed in their home cage for 5 min. Exploratory activity (total distance traveled) and scored behaviors (face washing, body grooming, wet dog shakes, and rearing) were observed in a familiar open field for 10 min. In animals receiving aCSF, there were no behavioral differences between restrained and unrestrained groups. For the entire 10-min observation period, animals receiving PACAP, whether restrained or not, displayed elevated face washing and body grooming with decreased locomotor activity and rearing. Among PACAP-injected animals, restrained animals displayed increased body grooming compared to unrestrained animals during the first 2 min in the open field suggesting a summation of the effects of peptide injection and stressor. The observed elevation in grooming is consistent with previous studies reporting similar increases following electrical-, NMDA-, CRH-, or stressor-induced activation of the PVN. Thus, at the level of the PVN, PACAP may act as an excitatory neuropeptide and augment behavioral responses to stressors.

  4. (/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.

  5. In vitro toxicity of mercury, cadmium, and arsenic to platelet aggregation: influence of adenylate cyclase and phosphodiesterase activity.

    PubMed

    Kumar, S V; Bhattacharya, S

    2000-01-01

    In vitro effect of mercury (Hg2+), cadmium (Cd2+), and arsenic (As3+) on adenylate cyclase (AC) and phosphodiesterase (PDE) activity in relation to platelet aggregation (PA) was studied in rats. Cd(2+) significantly elevated cAMP (p < 0.005) in a dose-dependent (5, 10 and 20 pmoles) manner while Hg(2+) and As(3+) significantly reduced the cAMP level (p < 0.01 and p < 0.005, respectively). Our studies further reveal that Hg21 and As(3+) inhibit AC and stimulate PDE activity with a concomitant increase in the rate of PA. On the other hand, Cd(2+) stimulates AC and inhibits PDE activity with a decrease in the rate of PA. The present investigation suggests that cellular cAMP is a regulatory molecule in the event of PA and the disruption of its homeostasis is directly correlated to xenobiotic effects on PA. It is concluded that other than divalent heavy metal cations, As(3+) appears to be one of the most toxic xenobiotics to platelet function.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. The Pseudomonas aeruginosa Chp Chemosensory System Regulates Intracellular cAMP Levels by Modulating Adenylate Cyclase Activity

    PubMed Central

    Fulcher, Nanette B.; Holliday, Phillip M.; Klem, Erich; Cann, Martin J.; Wolfgang, Matthew C.

    2010-01-01

    Summary Multiple virulence systems in the opportunistic pathogen Pseudomonas aeruginosa are regulated by the second messenger signaling molecule adenosine 3’, 5’-cyclic monophosphate (cAMP). Production of cAMP by the putative adenylate cyclase enzyme CyaB represents a critical control point for virulence gene regulation. To identify regulators of CyaB, we screened a transposon insertion library for mutants with reduced intracellular cAMP. The majority of insertions resulting in reduced cAMP mapped to the Chp gene cluster encoding a putative chemotaxis-like chemosensory system. Further genetic analysis of the Chp system revealed that it has both positive and negative effects on intracellular cAMP and that it regulates cAMP levels by modulating CyaB activity. The Chp system was previously implicated in the production and function of type IV pili (TFP). Given that cAMP and the cAMP-dependent transcriptional regulator Vfr control TFP biogenesis gene expression, we explored the relationship between cAMP, the Chp system and TFP regulation. We discovered that the Chp system controls TFP production through modulation of cAMP while control of TFP-dependent twitching motility is cAMP-independent. Overall, our data define a novel function for a chemotaxis-like system in controlling cAMP production and establish a regulatory link between the Chp system, TFP and other cAMP-dependent virulence systems. PMID:20345659

  12. Urinary Bladder Dysfunction and Altered Somatic Sensitivity in Pituitary Adenylate Cyclase Activating Polypeptide Knockout (PACAP−/−) Mice

    PubMed Central

    May, Victor; Vizzard, Margaret A.

    2010-01-01

    Purpose Pituitary adenylate cyclase activating polypeptide (PACAP) and receptors are expressed in micturition pathways. Studies demonstrated roles for PACAP in detrusor smooth muscle contraction, facilitating ATP release from urothelium and PACAP antagonism reduced cyclophosphamide-induced bladder hyperreflexia. Materials and Methods PACAP contributions to micturition and somatic sensation were studied in PACAP knockout (PACAP−/−), littermate heterozygote (PACAP+/−) and wildtype (WT) mice using conscious cystometry with continuous intravesical saline or acetic acid (AA; 0.5%) instillation, urination patterns, somatic sensitivity testing of hindpaw and pelvic region with calibrated von Frey filaments and morphological assessments of urinary bladder. Results PACAP−/− mice exhibit increased bladder mass with fewer but larger urine spots. In PACAP−/− mice, the lamina propria and detrusor smooth muscle are significantly thicker whereas the urothelium is unchanged. PACAP−/− mice exhibit increased bladder capacity, void volume (VV) and longer intercontraction interval (ICI) with significantly increased detrusor contraction duration and large residual volume. WT mice respond to AA (0.5%) with a reduction in VV and a decreased ICI whereas PACAP+/− and PACAP−/− mice do not respond. PACAP−/− mice are less responsive to somatic stimulation. PACAP+/− also exhibit bladder dysfunction and somatic and visceral sensory abnormalities but to a lesser degree. Conclusions PACAP gene disruption contributes to changes in bladder morphology, bladder function and somatic and visceral hypoalgesia. PMID:20022034

  13. 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.

  14. Pituitary adenylate cyclase activating peptide (PACAP) immunoreactivity and mRNA expression in the duck gastrointestinal tract.

    PubMed

    Mirabella, N; Squillacioti, C; Colitti, M; Germano, G; Pelagalli, A; Paino, G

    2002-06-01

    The presence and distribution of pituitary adenylate cyclase activating peptide (PACAP) immunoreactivity were studied in the duck gastrointestinal tract using immunohistochemistry and radioimmunoassays. Expression and distribution of PACAP mRNA were also studied using reverse transcriptase polymerase chain reaction (RT-PCR) and hybridization techniques. In addition, a partial coding sequence (cds) of the duck growth hormone-releasing hormone (GRF)/PACAP gene was identified. The presence of both PACAP-38 and PACAP-27 was demonstrated, the former being the predominant form. PACAP immunoreactivity was found in neurons and fibers of the enteric nervous system (ENS), in endocrine cells and in the gut associated lymphoid tissue (GALT). Double immunostaining showed that PACAP is almost completely colocalized with vasoactive intestinal peptide (VIP) in the ENS. Moreover, PACAP was also found in nitric oxide synthase (NOS)-containing neurons and nerve fibers. Radioimmunoassay (RIA) performed on denervated gut showed that more than one-half of the duodenal PACAP is extrinsic in origin. RT-PCR, Northern blot analysis and in situ hybridization confirmed the immunohistochemical data. The findings of the present study suggest that, in birds, PACAP may have multiple roles in regulating gastrointestinal functions.

  15. The Pseudomonas aeruginosa Chp chemosensory system regulates intracellular cAMP levels by modulating adenylate cyclase activity.

    PubMed

    Fulcher, Nanette B; Holliday, Phillip M; Klem, Erich; Cann, Martin J; Wolfgang, Matthew C

    2010-05-01

    Multiple virulence systems in the opportunistic pathogen Pseudomonas aeruginosa are regulated by the second messenger signalling molecule adenosine 3', 5'-cyclic monophosphate (cAMP). Production of cAMP by the putative adenylate cyclase enzyme CyaB represents a critical control point for virulence gene regulation. To identify regulators of CyaB, we screened a transposon insertion library for mutants with reduced intracellular cAMP. The majority of insertions resulting in reduced cAMP mapped to the Chp gene cluster encoding a putative chemotaxis-like chemosensory system. Further genetic analysis of the Chp system revealed that it has both positive and negative effects on intracellular cAMP and that it regulates cAMP levels by modulating CyaB activity. The Chp system was previously implicated in the production and function of type IV pili (TFP). Given that cAMP and the cAMP-dependent transcriptional regulator Vfr control TFP biogenesis gene expression, we explored the relationship between cAMP, the Chp system and TFP regulation. We discovered that the Chp system controls TFP production through modulation of cAMP while control of TFP-dependent twitching motility is cAMP-independent. Overall, our data define a novel function for a chemotaxis-like system in controlling cAMP production and establish a regulatory link between the Chp system, TFP and other cAMP-dependent virulence systems.

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. Effect of lipopolysaccharide on the transport of pituitary adenylate cyclase activating polypeptide across the blood-brain barrier.

    PubMed

    Nonaka, Naoko; Shioda, Seji; Banks, William A

    2005-01-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) has neuroprotective effects against ischemia, even when given by intravenous (iv) administration 24 h after stroke. Transport of PACAP across the blood-brain barrier (BBB) by peptide transport system (PTS)-6 underlies its effectiveness after iv administration. However, PACAP transport is modified after central nervous system (CNS) injury, raising the question of whether cytokines or BBB disruption affects PTS-6 activity. Lipopolysaccharide (LPS) is derived from bacterial cell walls and affects the passage of other proteins across the BBB through its release of cytokines and disruption of the BBB. Here, we examined by several methods the transport of radioactively labeled PACAP (I-PACAP) across the BBB after intraperitoneal (ip) injection of LPS. After three doses of LPS, studies at a single time point found a differential effect of LPS on the brain/serum ratio for I-PACAP and radioactively labeled albumin (I-Albumin). Whereas LPS increased the ratio for I-Albumin, demonstrating BBB disruption, it decreased the ratio for I-PACAP. Multiple-time regression analysis, capillary depletion, and brain perfusion showed that this decrease was fully explained by a decrease in the initial, reversible binding of I-PACAP to brain endothelium, while the rate of transport of PACAP into the brain was not altered. These methods also showed that the LPS-treated mice were volume contracted. This volume contraction concentrated the amount of I-PACAP in the blood and so increased the amount of I-PACAP presented to the BBB. Lack of change in transport rate combined with volume contraction resulted in a net increase of about 30% of the iv dose of I-PACAP entering the brain. LPS did not alter the efflux of I-PACAP from the CNS. In conclusion, PTS-6 remains active and should be able to deliver therapeutic amounts of PACAP to the CNS in brain injuries involving cytokine release and BBB disruption.

  2. 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. Copyright © 2010 Elsevier Inc. All rights reserved.

  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 Disrupts Motivation, Social Interaction, and Attention in Male Sprague Dawley Rats.

    PubMed

    Donahue, Rachel J; Venkataraman, Archana; Carroll, F Ivy; Meloni, Edward G; Carlezon, William A

    2016-12-15

    Severe or prolonged stress can trigger psychiatric illnesses including mood and anxiety disorders. Recent work indicates that pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in regulating stress effects. In rodents, exogenous PACAP administration can produce persistent elevations in the acoustic startle response, which may reflect anxiety-like signs including hypervigilance. We investigated whether PACAP causes acute or persistent alterations in behaviors that reflect other core features of mood and anxiety disorders (motivation, social interaction, and attention). Using male Sprague Dawley rats, we examined if PACAP (.25-1.0 µg, intracerebroventricular infusion) affects motivation as measured in the intracranial self-stimulation test. We also examined if PACAP alters interactions with a conspecific in the social interaction test. Finally, we examined if PACAP affects performance in the 5-choice serial reaction time task, which quantifies attention and error processing. Dose-dependent disruptions in motivation, social interaction, and attention were produced by PACAP, as reflected by increases in reward thresholds, decreases in social behaviors, and decreases in correct responses and alterations in posterror accuracy. Behavior normalized quickly in the intracranial self-stimulation and 5-choice serial reaction time task tests but remained dysregulated in the social interaction test. Effects on attention were attenuated by the corticotropin-releasing factor receptor-1 antagonist antalarmin but not the κ opioid receptor antagonist JDTic. Our findings suggest that PACAP affects numerous domains often dysregulated in mood and anxiety disorders, but that individual signs depend on brain substrates that are at least partially independent. This work may help to devise therapeutics that mitigate specific signs of these disorders. Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  5. 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.

  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. Targeted Pituitary Overexpression of Pituitary Adenylate-Cyclase Activating Polypeptide Alters Postnatal Sexual Maturation in Male Mice

    PubMed Central

    Yang, Rong Q.; Winters, Stephen J.

    2012-01-01

    The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) is present in high concentrations within the hypothalamus, suggesting that it may be a hypophysiotropic factor, whereas pituitary expression suggests a paracrine function. PACAP stimulates gonadotropin secretion and enhances GnRH responsiveness. PACAP increases gonadotropin α-subunit (αGSU), lengthens LHβ, but reduces FSHβ mRNA levels in adult pituitary cell cultures in part by increasing follistatin. PACAP stimulates LH secretion in rats; however, acceptance of PACAP as a regulator of reproduction has been limited by a paucity of in vivo studies. We created a transgenic mouse model of pituitary PACAP overexpression using the αGSU subunit promoter. Real-time PCR was used to evaluate PACAP, follistatin, GnRH receptor, and the gonadotropin subunit mRNA in male transgenic and wild-type mice of various ages. Transgenic mice had greater than 1000-fold higher levels of pituitary PACAP mRNA; and immunocytochemistry, Western blot, and ELISA analyses confirmed high peptide levels. FSH, LH, and testosterone levels were significantly suppressed, and the timing of puberty was substantially delayed in PACAP transgenic mice in which gonadotropin subunit and GnRH receptor mRNA levels were reduced and pituitary follistatin expression was increased. Microarray analyses revealed 1229 of 45102 probes were significantly (P < 0.01) different in pituitaries from PACAP transgenic mice, of which 83 genes were at least 2-fold different. Genes involved in small molecule biochemistry, cancer, and reproductive system diseases were the top associated networks. The GnRH signaling pathway was the top canonical pathway affected by pituitary PACAP excess. These experiments provide the first evidence that PACAP affects gonadotropin expression and sexual maturation in vivo. PMID:22315445

  8. 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.

  9. 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.

  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. 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.

  12. 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

  13. Characterization of adenylate cyclase toxin from a mutant of Bordetella pertussis defective in the activator gene, cyaC.

    PubMed

    Hewlett, E L; Gray, M C; Ehrmann, I E; Maloney, N J; Otero, A S; Gray, L; Allietta, M; Szabo, G; Weiss, A A; Barry, E M

    1993-04-15

    Bordetella pertussis adenylate cyclase (AC) toxin has the abilities to 1) enter target cells where it catalyzes cyclic AMP production and 2) lyse sheep erythrocytes, and these abilities require post-translational modification by the product of an accessory gene cyaC (Barry, E. M., Weiss, A. A., Ehrmann, E. E., Gray, M. C., Hewlett, E. L., and Goodwin, M. St. M. (1991) J. Bacteriol. 173, 720-726). In the present study, AC toxin has been purified from an organism with a mutation in cyaC, BPDE386, and evaluated for its physical and functional properties in order to determine the basis for its lack of toxin and hemolytic activities. AC toxin from BPDE386 is indistinguishable from wild-type toxin in enzymatic activity, migration on SDS-polyacrylamide gel electrophoresis, ability to bind calcium, and calcium-dependent conformational change. Although unable to elicit cAMP accumulation, AC toxin from BPDE386 exhibits binding to the surface of Jurkat cells which is comparable to that of wild-type toxin. This target cell interaction is qualitatively different, however, in that 99% of the mutant toxin remains sensitive to trypsin, whereas approximately 20% of cell-associated wild-type toxin enters a trypsin-resistant compartment. To evaluate the ability of this mutant AC toxin to function at its intracellular site of action, the cAMP-stimulated L-type calcium current in frog atrial myocytes was used. Extracellular addition of wild-type toxin results in cAMP-dependent events that include activation of calcium channels and enhancement of calcium current. In contrast, there is no response to externally applied toxin from BPDE386. When injected into the cell interior, however, the AC toxin from BPDE386 is able to produce increases in the calcium current comparable to those observed with wild-type toxin. Although AC toxin from BPDE386 is unaffected in its enzymatic activity, calcium binding, and calcium-dependent conformational change, the mutation in cyaC does result in a toxin

  14. 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.

  15. 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.

  16. 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.

  17. Independent sensitization of β-adrenoceptors and adenylate cyclase in acute myocardial ischaemia

    PubMed Central

    Strasser, R. H.; Marquetant, R.; Kübler, W.

    1990-01-01

    1 Acute myocardial ischaemia provokes sensitization of the adenylate cyclase system. This sensitization could be differentiated in a receptor-linked and an enzyme-linked sensitization. The increase in the number of β-adrenoceptors in the plasma membranes was observed already after 15 min of global ischaemia (50 ± 2 to 67 ± 6 fmol mg-1 protein) and persisted after 50 min of ischaemia. The maximally isoprenaline-stimulated adenylate cyclase activity rose from 66 ± 7 to 100 ± 10 pmol cAMP min-1 mg-1 protein after 15 min of global ischaemia indicating the receptor-mediated sensitization of the β-adrenergic system. However, after 50 min of ischaemia the isoprenaline-stimulated adenylate cyclase was reduced by about 50% despite the continuous increase of β-adrenoceptors in the plasma membranes. 2 Additionally direct stimulation of the adenylate cyclase by forskolin revealed an increased enzyme activity after 15 min of global ischaemia (300 ± 20 vs 378 ± 25 pmol cAMP min-1 mg-1). Prolonged periods of ischaemia, however, caused a decline of the total adenylate cyclase activity (232 ± 24 pmol cAMP min-1 mg-1 protein). This demonstrates an enzyme-specific sensitization of the adenylate cyclase, which in contrast to the rise in β-adrenoceptors is only transient. This enzyme-specific sensitization or the late inactivation of the enzyme occur independently of receptor activation and cannot be prevented by β-adrenoceptor blockade (10-6 M alprenolol) prior to the ischaemic insult. For the first time it could be demonstrated that the enzyme-linked sensitization is carried by the adenylate cyclase even after partial purification of the enzyme including solubilization and wheatgerm affinity chromatography. These data may suggest an ischaemia-induced covalent modification of the adenylate cyclase. The enzyme-linked sensitization and the late inactivation of the enzyme do not occur after cyanide perfusion demonstrating that energy depletion is not solely responsible for

  18. 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

  19. 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.

  20. Identification of a haem domain in human soluble adenylate cyclase

    PubMed Central

    Middelhaufe, Sabine; Leipelt, Martina; Levin, Lonny R.; Buck, Jochen; Steegborn, Clemens

    2012-01-01

    The second messengers cAMP and cGMP mediate a multitude of physiological processes. In mammals, these cyclic nucleotides are formed by related Class III nucleotidyl cyclases, and both ACs (adenylate cyclases) and GCs (guanylate cyclases) comprise transmembrane receptors as well as soluble isoforms. Whereas sGC (soluble GC) has a well-characterized regulatory HD (haem domain) that acts as a receptor for the activator NO (nitric oxide), very little is known about the regulatory domains of the ubiquitous signalling enzyme sAC (soluble AC). In the present study, we identify a unique type of HD as a regulatory domain in sAC. The sAC-HD (sAC haem domain) forms a larger oligomer and binds, non-covalently, one haem cofactor per monomer. Spectral analyses and mutagenesis reveal a 6-fold co-ordinated haem iron atom, probably with non-typical axial ligands, which can bind both NO and CO (carbon monoxide). Splice variants of sAC comprising this domain are expressed in testis and skeletal muscle, and the HD displays an activating effect on the sAC catalytic core. Our results reveal a novel mechanism for regulation of cAMP signalling and suggest a need for reanalysis of previous studies on mechanisms of haem ligand effects on cyclic nucleotide signalling, particularly in testis and skeletal muscle. PMID:22775536

  1. 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.

  2. 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

  3. [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.

  4. 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

  5. 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).

  6. 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.

  7. 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.

  8. 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

  9. 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.

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

    PubMed Central

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

    2015-01-01

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

  11. 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.

  12. 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.

  13. Globin-coupled heme containing oxygen sensor soluble adenylate cyclase in Leishmania prevents cell death during hypoxia.

    PubMed

    Sen Santara, Sumit; Roy, Jayasree; Mukherjee, Supratim; Bose, Moumita; Saha, Rina; Adak, Subrata

    2013-10-15

    Globin and adenylate cyclase play individually numerous crucial roles in eukaryotic organisms. Comparison of the amino acid sequences of globins and adenylate cyclase from prokaryotic to eukaryotic organisms suggests that they share an early common ancestor, even though these proteins execute different functions in these two kingdoms. The latest studies of biological signaling molecules in both prokaryotic and eukaryotic organisms have discovered a new class of heme-containing proteins that act as sensors. The protein of the globin family is still unknown in the trypanosomatid parasites, Trypanosome and Leishmania. In addition, globin-coupled heme containing adenylate cyclase is undescribed in the literature. Here we report a globin-coupled heme containing adenylate cyclase (HemAC-Lm) in the unicellular eukaryotic organism Leishmania. The protein exhibits spectral properties similar to neuroglobin and cytoglobin. Localization studies and activity measurements demonstrate that the protein is present in cytosol and oxygen directly stimulates adenylate cyclase activity in vivo and in vitro. Gene knockdown and overexpression studies suggest that O2-dependent cAMP signaling via protein kinase A plays a fundamental role in cell survival through suppression of oxidative stress under hypoxia. In addition, the enzyme-dependent cAMP generation shows a stimulatory as well as inhibitory role in cell proliferation of Leishmania promastigotes during normoxia. Our work begins to clarify how O2-dependent cAMP generation by adenylate cyclase is likely to function in cellular adaptability under various O2 tensions.

  14. Properties of Adenyl Cyclase from Human Jejunal Mucosa during Naturally Acquired Cholera and Convalescence

    PubMed Central

    Chen, Lincoln C.; Rohde, Jon E.; Sharp, Geoffrey W. G.

    1972-01-01

    The enterotoxin of Vibrio cholerae causes copious fluid production throughout the lenght of the small intestine. As this is thought to be mediated by stimulation of adenyl cyclase, a study has been made of the activity and properties of this enzyme in jejunal biopsy tissue taken from patients during the diarrheal phase of cholera and after recovery. Adenyl cyclase activity during cholera was increased more than twofold relative to the enzyme in convalescence. Under both conditions stimulation by prostaglandin E1 (PGE1) and by fluoride was observed. The responsiveness to PGE1 was not altered in cholera; the total activity of the fluoride-stimulated enzyme was similar, a finding that suggests cholera toxin stimulates pre-existing enzyme in the intestinal cell. The enzymes during cholera and convalescence were similar in all other properties examined. Optimal Mg++ concentration was 10 mM; Mn++ at 5 mM stimulated the enzyme but could not replace Mg++ except in the presence of 10 mM fluoride. Calcium was markedly inhibitory at concentrations greater than 10-4 M. The pH optimum was 7.5 and the Michaelis constant (Km) for ATP concentration approximated 10-4 M. Thus the interaction of cholera toxin with human intestinal adenyl cyclase does not alter the basic properties of the enzyme. When biopsy specimens were maintained intact in oxygenated Ringer's solution at 0°C, no loss of activity was observed at 1½ and 3 hr. In contrast, when the cells were homogenized, rapid loss of activity, with a half-life of 90 min was seen even at 0°C. Consequently for comparative assays of human jejunal adenyl cyclase, strict control of the experimental conditions is required. It was under such conditions that a twofold increase in basal adenyl cyclase activity during cholera was observed. Images PMID:4335441

  15. 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.

  16. Dependence of the hormonal stimulation of adenylate cyclase on the fraction of the plasma membrane accessible for lateral displacement of proteins of the adenylate cyclase complex

    SciTech Connect

    Kazarov, A.R.; Rozenkrants, A.A.; Sobolev, A.S.

    1986-09-10

    Hormonal activation of the adenylate cyclase complex is associated with lateral displacement in the membrane of the proteins that constitute this complex. In this work an experimental investigation was made of the changes in the interaction of the proteins of the adenylate cyclase complex with the changing fraction of fluid lipids in the cell membrane. A decrease in the fraction of fluid lipids of rat reticulocyte membranes led to a decrease (all the way down to a total suppression) of the interaction of the ..beta..-adrenoreceptors with the regulatory N-proteins. The interaction of the N-proteins with the catalytic proteins was also suppressed. On the other hand, an increase in the fraction of fluid lipids led to more effective interaction. It was shown that in this case the functional intactness of the interacting proteins is unimpaired. An analysis of the results obtained, performed on the basis of the percolation theory, suggests the conclusion that the hormonal stimulation of adenylate cyclase depends on the fraction of fluid lipids in the membrane, and the proteins are displaced during interaction over distances comparable with the size of the membrane itself. It was also shown that characteristic activity of the ..beta..-agonist 1-isoproterenol varies from 1.0 to 0, depending on the fraction of fluid lipids in the membrane. The data obtained suggest that in the absence of guanylic nucleotides in the membrane in vitro there are no preexisting complexes with a high affinity for the agonist.

  17. [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.

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. Bordetella pertussis Commits Human Dendritic Cells to Promote a Th1/Th17 Response through the Activity of Adenylate Cyclase Toxin and MAPK-Pathways

    PubMed Central

    Palazzo, Raffaella; Nasso, Maria; Cheung, Gordon Yiu Chong; Coote, John Graham; Ausiello, Clara Maria

    2010-01-01

    The complex pathology of B. pertussis infection is due to multiple virulence factors having disparate effects on different cell types. We focused our investigation on the ability of B. pertussis to modulate host immunity, in particular on the role played by adenylate cyclase toxin (CyaA), an important virulence factor of B. pertussis. As a tool, we used human monocyte derived dendritic cells (MDDC), an ex vivo model useful for the evaluation of the regulatory potential of DC on T cell immune responses. The work compared MDDC functions after encounter with wild-type B. pertussis (BpWT) or a mutant lacking CyaA (BpCyaA−), or the BpCyaA− strain supplemented with either the fully functional CyaA or a derivative, CyaA*, lacking adenylate cyclase activity. As a first step, MDDC maturation, cytokine production, and modulation of T helper cell polarization were evaluated. As a second step, engagement of Toll-like receptors (TLR) 2 and TLR4 by B. pertussis and the signaling events connected to this were analyzed. These approaches allowed us to demonstrate that CyaA expressed by B. pertussis strongly interferes with DC functions, by reducing the expression of phenotypic markers and immunomodulatory cytokines, and blocking IL-12p70 production. B. pertussis-treated MDDC promoted a mixed Th1/Th17 polarization, and the activity of CyaA altered the Th1/Th17 balance, enhancing Th17 and limiting Th1 expansion. We also demonstrated that Th1 effectors are induced by B. pertussis-MDDC in the absence of IL-12p70 through an ERK1/2 dependent mechanism, and that p38 MAPK is essential for MDDC-driven Th17 expansion. The data suggest that CyaA mediates an escape strategy for the bacterium, since it reduces Th1 immunity and increases Th17 responses thought to be responsible, when the response is exacerbated, for enhanced lung inflammation and injury. PMID:20090944

  3. Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells

    PubMed Central

    Martín, César; Etxaniz, Asier; Uribe, Kepa B.; Etxebarria, Aitor; González-Bullón, David; Arlucea, Jon; Goñi, Félix M.; Aréchaga, Juan; Ostolaza, Helena

    2015-01-01

    Bordetella pertussis causes whooping cough, a respiratory infectious disease that is the fifth largest cause of vaccine-preventable death in infants. Though historically considered an extracellular pathogen, this bacterium has been detected both in vitro and in vivo inside phagocytic and non-phagocytic cells. However the precise mechanism used by B. pertussis for cell entry, or the putative bacterial factors involved, are not fully elucidated. Here we find that adenylate cyclase toxin (ACT), one of the important toxins of B. pertussis, is sufficient to promote bacterial internalisation into non-phagocytic cells. After characterization of the entry route we show that uptake of “toxin-coated bacteria” proceeds via a clathrin-independent, caveolae-dependent entry pathway, allowing the internalised bacteria to survive within the cells. Intracellular bacteria were found inside non-acidic endosomes with high sphingomyelin and cholesterol content, or “free” in the cytosol of the invaded cells, suggesting that the ACT-induced bacterial uptake may not proceed through formation of late endolysosomes. Activation of Tyr kinases and toxin-induced Ca2+-influx are essential for the entry process. We hypothesize that B. pertussis might use ACT to activate the endocytic machinery of non-phagocytic cells and gain entry into these cells, in this way evading the host immune system. PMID:26346097

  4. 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

  5. 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

  6. Behavioral stimulation without alteration of beta and 5-HT receptors and adenylate cyclase activity in rat brain after chronic sertraline administration.

    PubMed

    Tadokoro, C; Kiuchi, Y; Yamazaki, Y; Nara, K; Oguchi, K; Kamijima, K

    1997-03-01

    Effects of chronic treatment with selective 5-HT reuptake inhibitors (SSRIs) on the monoaminergic functions have not been much investigated in compared with tricyclic antidepressants. Therefore, we compared the effects of 3-week treatment with sertraline, a potent SSRI, to those of imipramine (10 mg/kg, IP, twice a day), on monoamine receptors and adenylate cyclase (AC) activity in rat brain. Two-week treatment with both sertraline and imipramine reduced immobility in the water wheel test to the comparable extent. Sertraline treatment did not affect Kd and Bmax of [3H]CGP12177 and [3H]ketanserin bindings or cAMP, accumulation by norepinephrine, isoproternol, 5'-guanylylimidodiphosphate [Gpp(NH)p] and forskolin in the cortical membrane compared with vehicle-treated rats. On the other hand, imipramine treatment decreased Bmax of both bindings and norepinephrine- or isoproternol-stimulated cAMP accumulation. Treatment with either antidepressant induced no apparent changes in [3H]8-OH-DPAT [2-(N, N-dipropylamino)-8-hydroxy-1,2,3,4-tetrahydronaphthalene] binding in the hippocampal membrane. These results suggested that chronic treatment of sertraline induced little effect on monoamine receptors and AC activity in the brain and that the alteration of these functions may not be primarily involved in antidepressive effects of antidepressants, at least of SSRIs.

  7. Interaction of Gonadal Steroids and Gonadotropin-Releasing Hormone on Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and PACAP Receptor Expression in Cultured Rat Anterior Pituitary Cells

    PubMed Central

    Zheng, Weiming; Grafer, Constance M.

    2014-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are expressed in the hypothalamus, the gonadotrope cells of the anterior pituitary gland, and the gonads, forming an autocrine–paracrine system in these tissues. Within the pituitary, PACAP functions either alone or synergistically with gonadotropin-releasing hormone (GnRH) to stimulate gonadotropin gene expression and secretion. Our goal was to define the hormonal regulation of pituitary PACAP and PACAP receptor (PAC1) gene expression by dihydrotestosterone (DHT), estradiol, and progesterone alone or in conjunction with GnRH. Treatment of adult male rat pituitary cell cultures with DHT or progesterone augmented GnRH-mediated increase in PACAP messenger RNA (mRNA) levels, but neither had an effect when present alone. Conversely, estradiol treatment blunted PACAP gene expression but did not alter GnRH effects on PACAP expression. Expression of PACAP receptor mRNA was decreased by GnRH treatment, minimally increased by DHT treatment, but not altered by the addition of estradiol or progesterone. DHT and GnRH together blunted PACAP receptor gene expression. Taken together, these results suggest that the activity of the intrapituitary PACAP-PAC1 system is regulated via the complex interaction of gonadal steroids and hypothalamic GnRH. PMID:23690336

  8. Interictal plasma pituitary adenylate cyclase-activating polypeptide levels are decreased in migraineurs but remain unchanged in patients with tension-type headache.

    PubMed

    Han, Xun; Dong, Zhao; Hou, Lei; Wan, Dongjun; Chen, Min; Tang, Wenjing; Yu, Shengyuan

    2015-10-23

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is associated with migraine phase; however, whether PACAP levels could be used to distinguish between migraine and tension-type headache (TTH) remains unknown. We compared interictal plasma PACAP levels among healthy controls, migraineurs, and patients with TTH. Interictal plasma levels of PACAP were measured in 133 migraineurs, 106 patients with TTH, and 50 controls using enzyme-linked immunoassays. We further evaluated the relationships between interictal PACAP plasma concentrations and clinical parameters, such as headache severity, attack frequency, and duration. We found that migraineurs had significantly lower interictal plasma PACAP levels than patients with TTH and healthy controls. However, there were no significant differences between patients with TTH and healthy controls. Plasma PACAP levels were significantly lower in patients with episodic migraine (EM) than in patients with episodic tension-type headache (ETTH) and in patients with chronic migraine (CM) than in patients with chronic tension-type headache (CTTH). Interictal PACAP levels were negatively correlated with duration in the CM group. The results of this study demonstrated differences in interictal PACAP levels in migraine and TTH, suggesting that PACAP is involved in the pathogenesis of migraine rather than TTH. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Sudden infant death syndrome (SIDS) in African Americans: polymorphisms in the gene encoding the stress peptide pituitary adenylate cyclase-activating polypeptide (PACAP).

    PubMed

    Cummings, Kevin J; Klotz, Cherise; Liu, Wei-Qiao; Weese-Mayer, Debra E; Marazita, Mary L; Cooper, Margaret E; Berry-Kravis, Elizabeth M; Tobias, Rose; Goldie, Cameron; Bech-Hansen, N Torben; Wilson, Richard Ja

    2009-03-01

    Mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP) are prone to sudden death in the second post-natal week, having respiratory and metabolic disturbances reminiscent of the human Sudden Infant Death Syndrome (SIDS). Here we test the hypothesis that the human PACAP gene is a site of genetic variance associated with SIDS in a cohort of 92 victims and 92 matched controls. Using polymerase chain reaction and sequencing, we examined the PACAP gene in 92 SIDS cases (46 Caucasians and 46 African Americans) and 92 race- and gender-matched controls. We found no significant associations between PACAP and SIDS in Caucasians. However, in the African Americans, a non-synonymous single nucleotide polymorphism (i.e. an aspartic acid/glycine coding variant, rs2856966) within exon 2 of PACAP was significantly associated with SIDS (p = 0.004), as were haplotypes containing this polymorphism (p < 0.0001). Glycine was three times more likely at this location in the African-American SIDS victims (17 cases) than African-American controls (5 cases). These data are the first to suggest an association between a variant within the coding region of the PACAP gene and SIDS. Based on these findings, further investigations are warranted into the functional importance of PACAP signaling in neonatal survival and the role of PACAP-signaling abnormalities in SIDS.

  10. 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.

  11. 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.

  12. 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.

  13. Catecholamine-sensitive adenylate cyclase of caudate nucleus and cerebral cortex. Effects of guanine nucleotides.

    PubMed Central

    Sulakhe, P V; Leung, N L; Arbus, A T; Sulakhe, S J; Jan, S H; Narayanan, N

    1977-01-01

    1. GTP and GMP-P(NH)P (guanyl-5'-yl imidodiphosphate) were observed to increase the stimulation of neural adenylate cyclase by dopamine (3,4-dihydroxyphenethylamine) and noradrenaline. 2. GMP-P(NH)P had a biphasic effect on the enzyme activity. 3. Preincubation of membranes with GMP-P(NH)P activated the enzyme by a process dependent on time and temperature. Catecholamines increased the speed and the extent of this activation. 4. Membrane fractions contained high- and low-affinity sites for GMP-P(NH)P binding: this binding was due to protein(s) of the membrane preparations. 5. Low-affinity-site binding of GMP-P(NH)P appeared to be related to the stimulatory effect on the adenylate cyclase activity. PMID:18147

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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

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

    PubMed Central

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

    2015-01-01

    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. PMID:25649277

  4. 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

  5. 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.

  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. 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)

  8. Solution structure and mutational analysis of pituitary adenylate cyclase-activating polypeptide binding to the extracellular domain of PAC1-RS

    PubMed Central

    Sun, Chaohong; Song, Danying; Davis-Taber, Rachel A.; Barrett, Leo W.; Scott, Victoria E.; Richardson, Paul L.; Pereda-Lopez, Ana; Uchic, Marie E.; Solomon, Larry R.; Lake, Marc R.; Walter, Karl A.; Hajduk, Philip J.; Olejniczak, Edward T.

    2007-01-01

    The pituitary adenylate cyclase-activating polypeptide (PACAP) receptor is a class II G protein-coupled receptor that contributes to many different cellular functions including neurotransmission, neuronal survival, and synaptic plasticity. The solution structure of the potent antagonist PACAP (residues 6′–38′) complexed to the N-terminal extracellular (EC) domain of the human splice variant hPAC1-R-short (hPAC1-RS) was determined by NMR. The PACAP peptide adopts a helical conformation when bound to hPAC1-RS with a bend at residue A18′ and makes extensive hydrophobic and electrostatic interactions along the exposed β-sheet and interconnecting loops of the N-terminal EC domain. Mutagenesis data on both the peptide and the receptor delineate the critical interactions between the C terminus of the peptide and the C terminus of the EC domain that define the high affinity and specificity of hormone binding to hPAC1-RS. These results present a structural basis for hPAC1-RS selectivity for PACAP versus the vasoactive intestinal peptide and also differentiate PACAP residues involved in binding to the N-terminal extracellular domain versus other parts of the full-length hPAC1-RS receptor. The structural, mutational, and binding data are consistent with a model for peptide binding in which the C terminus of the peptide hormone interacts almost exclusively with the N-terminal EC domain, whereas the central region makes contacts to both the N-terminal and other extracellular parts of the receptor, ultimately positioning the N terminus of the peptide to contact the transmembrane region and result in receptor activation. PMID:17470806

  9. Pituitary adenylate cyclase-activating polypeptide enhances saliva secretion via direct binding to PACAP receptors of major salivary glands in mice.

    PubMed

    Matoba, Yuko; Nonaka, Naoko; Takagi, Yoshitoki; Imamura, Eisaku; Narukawa, Masayuki; Nakamachi, Tomoya; Shioda, Seiji; Banks, William A; Nakamura, Masanori

    2016-09-01

    Xerostomia, or dry mouth, is a common syndrome that is generally treated with artificial saliva; however, no other effective methods have yet been established. Saliva secretion is mainly under the control of the autonomic nervous system. Pituitary adenylate cyclase-activating polypeptide (PACAP) is recognized as a multifunctional neuropeptide in various organs. In this study, we examined the effect of PACAP on saliva secretion, and detected the distribution of the PACAP type 1 receptor (PAC1R) in major salivary glands, including the parotid, submandibular, and sublingual glands, in 9-week-old male C57BL/6 mice. Intranasal administration of PACAP 38 increased the amount of saliva secreted, which was not inhibited by atropine pretreatment. Immunohistochemical analysis showed that PAC1R was distributed in the three major salivary glands. In the parotid and sublingual glands, PAC1R was detected in striated duct cells, whereas in the submandibular gland, a strong PAC1R immunoreaction was detected in tall columnar epithelial cells in the granular ducts (i.e., pillar cells), as well as in some striated duct cells. PACAP significantly increased the concentration of epidermal growth factor in saliva. These results suggest that PACAP directly regulates saliva secretion by controlling the absorption activity in the ducts, and that pillar cells regulate the function of granular epithelial cells in the granular duct, such as the secretion of growth factors into the saliva. Collectively, these results suggest the possibility of PACAP as a new effective treatment of xerostomia. Anat Rec, 299:1293-1299, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  10. 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.

  11. 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.

  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. 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.

  14. 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

  15. 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.

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

    PubMed

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

    2016-02-01

    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 μg) or vehicle followed 30 min later by fear conditioning. Freezing was measured early (1, 4, and 7 days) 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). Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Pituitary adenylate cyclase activating polypeptide (PACAP) and its receptor 1 (PAC1) in the human infant brain and changes in the Sudden Infant Death Syndrome (SIDS).

    PubMed

    Huang, J; Waters, K A; Machaalani, R

    2017-07-01

    Pituitary adenylate cyclase activating polypeptide (PACAP) and its complementary receptor, PAC1, are crucial in central respiratory control. PACAP Knockout (KO) mice exhibit a SIDS-like phenotype, with an inability to overcome noxious insults, compression of baseline ventilation, and death in the early post-neonatal period. PAC1 KO demonstrate similar attributes to PACAP-null mice, but with the addition of increased pulmonary artery pressure, consequently leading to heart failure and death. This study establishes a detailed interpretation of the neuroanatomical distribution and localization of both PACAP and PAC1 in the human infant brainstem and hippocampus, to determine whether any changes in expression are evident in infants who died of Sudden Infant Death Syndrome (SIDS) and any relationships to risk factors of SIDS including smoke exposure and sleep related parameters. Immunohistochemistry for PACAP and PAC1 was performed on formalin fixed and paraffin embedded human infant brain tissue of SIDS (n=32) and non-SIDS (n=12). The highest expression of PACAP was found in the hypoglossal (XII) of the brainstem medulla and lowest expression in the subiculum of the hippocampus. Highest expression of PAC1 was also found in XII of the medulla and lowest in the midbrain dorsal raphe (MBDR) and inferior colliculus. SIDS compared to non-SIDS had higher PACAP in the MBDR (p<0.05) and lower PAC1 in the medulla arcuate nucleus (p<0.001). Correlations were found between PACAP and PAC1 with the risk factors of smoke exposure, bed sharing, upper respiratory tract infection (URTI) and seasonal temperatures. The findings of this study show for the first time that some abnormalities of the PACAP system are evident in the SIDS brain and could contribute to the mechanisms of infants succumbing to SIDS. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Correlation between oocyte number and follicular fluid concentration of pituitary adenylate cyclase-activating polypeptide (PACAP) in women after superovulation treatment.

    PubMed

    Koppan, M; Varnagy, A; Reglodi, D; Brubel, R; Nemeth, J; Tamas, A; Mark, L; Bodis, J

    2012-11-01

    Follicular growth, ovulation, and luteinization are influenced by interactions of peptide and steroid hormone-signaling cascades in the ovary. Pituitary adenylate cyclase-activating polypeptide (PACAP) plays an important role in the regulation of several endocrine processes and is present in ovarian follicular fluid (FF). However, little is known about PACAP in FF with regard to maturation, ovulation, fertilization, and successful pregnancy. The aim of this pilot study was to investigate whether there is a correlation between PACAP concentration in FF and ovarian response to superovulation treatment in infertile women, performed in volunteers (n = 132; aged between 20 and 35). After treatment, the number of harvested oocytes was recorded and PACAP immunoreactivity in FF was measured by radioimmunoassay. All the corresponding PACAP concentrations were below 290 fmol/ml in cases when the number of harvested oocytes exceeded 14 per patient, while in all cases above 290 fmol/ml, the number of oocytes was below 14. Using these cutoff values, we determined three study groups: high-PACAP concentration, high-oocyte number, and low-PACAP concentration-low-oocyte number groups. Median values of PACAP concentration in these groups were 411.2, 106.5, and 101.0 fmol/ml, respectively, while the median values of harvested oocytes were 5.5, 19.0, and 5.0, respectively. Differences were significant, indicating a correlation between concentration of PACAP in FF and the number of recruited oocytes. Higher concentrations of PACAP in FF might be associated with lower number of developing oocytes, while low concentrations of PACAP might correlate with a markedly higher number of ova retrieved, thus predicting a higher chance for ovarian hyperstimulation. Our present study is among the first few human clinical studies with direct conclusions drawn for possible clinical impact of PACAP.

  19. 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.

  20. 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).

  1. 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

  2. Hypothalamic and Brainstem Sources of Pituitary Adenylate Cyclase-Activating Polypeptide Nerve Fibers Innervating the Hypothalamic Paraventricular Nucleus in the Rat

    PubMed Central

    DAS, MAHASWETA; VIHLEN, CHRISTOPHER S.; LEGRADI, GABOR

    2007-01-01

    The hypothalamic paraventricular nucleus (PVN) coordinates major neuroendocrine and behavioral mechanisms, particularly responses to homeostatic challenges. Parvocellular and magnocellular PVN neurons are richly innervated by pituitary adenylate cyclase-activating polypeptide (PACAP) axons. Our recent functional observations have also suggested that PACAP may be an excitatory neuropeptide at the level of the PVN. Nevertheless, the exact localization of PACAP-producing neurons that project to the PVN is not understood. The present study examined the specific contribution of various brain areas sending PACAP innervation to the rat PVN by using iontophoretic microinjections of the retrograde neuroanatomical tracer cholera toxin B subunit (CTb). Retrograde transport was evaluated from hypothalamic and brainstem sections by using multiple labeling immunofluorescence for CTb and PACAP. PACAP-containing cell groups were found to be retrogradely labeled from the PVN in the median preoptic nucleus; preoptic and lateral hypothalamic areas; arcuate, dorsomedial, ventromedial, and supramammillary nuclei; ventrolateral midbrain periaqueductal gray; rostral and midlevel ventrolateral medulla, including the C1 catecholamine cell group; nucleus of the solitary tract; and dorsal motor nucleus of vagus. Minor PACAP projections with scattered double-labeled neurons originated from the parabrachial nucleus, pericoeruleus area, and caudal regions of the nucleus of the solitary tract and ventrolateral medulla. These observations indicate a multisite origin of PACAP innervation to the PVN and provide a strong chemical neuroanatomical foundation for interaction between PACAP and its potential target neurons in the PVN, such as parvocellular CRH neurons, controlling physiologic responses to stressful challenges and other neuroendocrine or preautonomic PVN neurons. PMID:17154257

  3. The activity of dopamine-stimulated adenylate cyclase from rat brain striatum is modulated by temperature and the bilayer-fluidizing agent, benzyl alcohol

    PubMed Central

    Needham, Lindsey; Houslay, Miles D.

    1982-01-01

    Benzyl alcohol achieved a marked activation of the adenylate cyclase activity in a partially purified membrane preparation from rat brain striata, although inhibition resulted at high concentrations. The degree of activation observed depended on the ligand used to stimulate the enzyme, with that observed in the presence of guanosine 5′-[β,γ-imido]triphosphate (p[NH]ppG) (5.8-fold)>dopamine+p[NH]ppG (5-fold)> GTP (3-fold)>dopamine+high GTP (2.25-fold)>dopamine (+low GTP)=basal (+low GTP) (1.7-fold). The differences in the concentration-dependence of both the activation and inhibition of dopamine-stimulated and basal activities of the enzyme meant that increasing benzyl alcohol concentrations caused a net elevation in the fold-stimulation of the basal activity by dopamine. Arrhenius plots of p[NH]ppG-, GTP-, fluoride-, dopamine-plus-high GTP- and dopamine-plus-p[NH]ppG-stimulated activities all exhibited a single break occurring at around 22°C. This break point was decreased to around 13°C when 50mm-benzyl alcohol was added to the assays. In the presence of dopamine (+low GTP), Arrhenius plots exhibited two distinct breaks, one at around 21°C and the other at around 11°C. When benzyl alcohol (50mm) was added to these assays of dopamine (+low GTP)-stimulated activity, a single break at around 14°C was observed. For the basal activity the Arrhenius plot exhibited a single break at around 15°C both in the presence and in the absence of 50mm-benzyl alcohol. It is suggested that the enzyme is activated by productive collisions between independent mobile entities and that the activity of the enzyme may be regulated by changes in membrane fluidity. The breaks in the Arrhenius plots of all of the ligand-stimulated activities, but not the basal activity, are attributed to lipid-phase separations occurring in either the inner or the outer halves of the bilayer. PMID:7126197

  4. Pituitary adenylate cyclase-activating polypeptide (PACAP) mimics neuroendocrine and behavioral manifestations of stress: Evidence for PKA-mediated expression of the corticotropin-releasing hormone (CRH) gene.

    PubMed

    Agarwal, Anika; Halvorson, Lisa M; Legradi, Gabor

    2005-07-29

    The physiologic response to stress is highly dependent on the activation of corticotropin-releasing hormone (CRH) neurons by various neurotransmitters. A particularly rich innervation of hypophysiotropic CRH neurons has been detected by nerve fibers containing the neuropeptide PACAP, a potent activator of the cAMP-protein kinase A (PKA) system. Intracerebroventricular (icv) injections of PACAP also elevate steady-state CRH mRNA levels in the paraventricular nucleus (PVN), but it is not known whether PACAP effects can be associated with acute stress responses. Likewise, in cell culture studies, pharmacologic activation of the PKA system has stimulated CRH gene promoter activity through an identified cAMP response element (CRE); however, a direct link between PACAP and CRH promoter activity has not been established. In our present study, icv injection of 150 or 300 pmol PACAP resulted in robust phosphorylation of the transcription factor CREB in the majority of PVN CRH neurons at 15 to 30 min post-injection and induced nuclear Fos labeling at 90 min. Simultaneously, plasma corticosterone concentrations were elevated in PACAP-injected animals, and significant increases were observed in face washing, body grooming, rearing and wet-dog shakes behaviors. We investigated the effect of PACAP on human CRH promoter activity in alphaT3-1 cells, a PACAP-receptor expressing cell line. Cells were transiently transfected with a chloramphenicol acetyltransferase (CAT) reporter vector containing region - 663/+124 of the human CRH gene promoter then treated for with PACAP (100 nM) or with the adenylate cyclase activating agent, forskolin (2.5 muM). Both PACAP and forskolin significantly increased wild-type hCRH promoter activity relative to vehicle controls. The PACAP response was abolished in the CRE-mutant construct. Pretreatment of transfected cells with the PKA blocker, H-89, completely prevented both PACAP- and forskolin-induced increases in CRH promoter activity. Furthermore

  5. 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.

  6. Serotonin-Sensitive Adenylate Cyclase in Neural Tissue and Its Similarity to the Serotonin Receptor: A Possible Site of Action of Lysergic Acid Diethylamide

    PubMed Central

    Nathanson, James A.; Greengard, Paul

    1974-01-01

    An adenylate cyclase (EC 4.6.1.1) that is activated specifically by low concentrations of serotonin has been identified in homogenates of the thoracic ganglia of an insect nervous system. The activation of this enzyme by serotonin was selectively inhibited by extremely low concentrations of D-lysergic acid diethylamide (LSD), 2-bromo-LSD, and cyproheptadine, agents which are known to block certain serotonin receptors in vivo. The inhibition was competitive with respect to serotonin, and the calculated inhibitory constant of LSD for this serotonin-sensitive adenylate cyclase was 5 nM. The data are consistent with a model in which the serotonin receptor of neural tissue is intimately associated with a serotonin-sensitive adenylate cyclase which mediates serotonergic neurotransmission. The results are also compatible with the possibility that some of the physiological effects of LSD may be mediated through interaction with serotonin-sensitive adenylate cyclase. PMID:4595572

  7. 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

  8. 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.

  9. 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.

  10. 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.

  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. 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.

  13. 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.

  14. 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

  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. Ex vivo and in vivo studies of CME-1, a novel polysaccharide purified from the mycelia of Cordyceps sinensis that inhibits human platelet activation by activating adenylate cyclase/cyclic AMP.

    PubMed

    Lu, Wan-Jung; Chang, Nen-Chung; Jayakumar, Thanasekaran; Liao, Jiun-Cheng; Lin, Mei-Jiun; Wang, Shwu-Huey; Chou, Duen-Suey; Thomas, Philip Aloysius; Sheu, Joen-Rong

    2014-12-01

    CME-1, a novel water-soluble polysaccharide, was purified from the mycelia of Cordyceps sinensis, and its chemical structure was characterized to contain mannose and galactose in a ratio of 4:6 (27.6 kDa). CME-1 was originally observed to exert a potent inhibitory effect on tumor migration and a cytoprotective effect against oxidative stress. Activation of platelets caused by arterial thrombosis is relevant to various cardiovascular diseases (CVDs). However, no data are available concerning the effects of CME-1 on platelet activation. Hence, the purpose of this study was to examine the ex vivo and in vivo antithrombotic effects of CME-1 and its possible mechanisms in platelet activation. The aggregometry, immunoblotting, flow cytometric analysis and platelet functional analysis were used in this study. CME-1 (2.3-7.6 μM) exhibited highly potent activity in inhibiting human platelet aggregation when stimulated by collagen, thrombin, and arachidonic acid but not by U46619. CME-1 inhibited platelet activation accompanied by inhibiting Akt, mitogen-activated protein kinases (MAPKs), thromboxane B2 (TxB2) and hydroxyl radical (OH(●)) formation. However, CME-1 interrupted neither FITC-triflavin nor FITC-collagen binding to platelets. CME-1 markedly increased cyclic AMP levels, but not cyclic GMP levels, and stimulated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, but not ODQ, an inhibitor of guanylate cyclase, obviously reversed the CME-1-mediated effects on platelet aggregation and vasodilator-stimulated phosphoprotein (VASP), Akt, p38 MAPK phosphorylation, and TxB2 formation. CME-1 substantially prolonged the closure time of whole blood and the occlusion time of platelet plug formation. This study demonstrates for the first time that CME-1 exhibits highly potent antiplatelet activity that may initially activate adenylate cyclase/cyclic AMP and, subsequently, inhibit intracellular signals (such as Akt and

  17. Structure-Function Relationships Underlying the Capacity of Bordetella Adenylate Cyclase Toxin to Disarm Host Phagocytes.

    PubMed

    Novak, Jakub; Cerny, Ondrej; Osickova, Adriana; Linhartova, Irena; Masin, Jiri; Bumba, Ladislav; Sebo, Peter; Osicka, Radim

    2017-09-24

    Bordetellae, pathogenic to mammals, produce an immunomodulatory adenylate cyclase toxin-hemolysin (CyaA, ACT or AC-Hly) that enables them to overcome the innate immune defense of the host. CyaA subverts host phagocytic cells by an orchestrated action of its functional domains, where an extremely catalytically active adenylyl cyclase enzyme is delivered into phagocyte cytosol by a pore-forming repeat-in-toxin (RTX) cytolysin moiety. By targeting sentinel cells expressing the complement receptor 3, known as the CD11b/CD18 (αMβ₂) integrin, CyaA compromises the bactericidal functions of host phagocytes and supports infection of host airways by Bordetellae. Here, we review the state of knowledge on structural and functional aspects of CyaA toxin action, placing particular emphasis on signaling mechanisms by which the toxin-produced 3',5'-cyclic adenosine monophosphate (cAMP) subverts the physiology of phagocytic cells.

  18. 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.

  19. 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.

  20. 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.

  1. Supersensitivity of beta-adrenoceptor coupled adenylate cyclase in pulmonary tissue of the spontaneously hypertensive rat

    SciTech Connect

    Kamibayashi, C.; Ramanathan, S. )

    1989-01-01

    Basal adenylate cyclase activity was similar in plasma membranes prepared from the lungs of 12 week old spontaneously hypertensive rats (SHR) and normotensive Wister Kyoto rats (WKY). However, sensitivity to Gpp (NH)p, isoproterenol plus GTP or Gpp (NH)p was significantly greater in the SHR. Beta-receptor density measured by ({sup 3}H)DHA binding was unaltered. The dissociation constant, K{sub d}, revealed a significantly greater binding affinity of the radioligand in the SHR compared with the WKY. Activity of G{sub s} was assessed by complementing S49 cyc{sup {minus}} acceptor membranes with lung cholate extract. Basal activity of the reconstituted system was decreased 43% in the SHR. However, sensitivity to NaF, Gpp(NH)p, and isoproterenol plus Gpp(NH)p was significantly elevated. These data suggest that desensitization of the adenylate cyclase complex is not a generalized response to chronic hypertension. A tissue specific increase in sympathetic drive appears to be responsible for the lowered concentration of cardiac beta-adrenoceptors in the SHR. In contrast, both indirect and direct evidence indicate an enhanced functional sensitivity of pulmonary G{sub s} in the hypertensive rats.

  2. 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

  3. 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.

  4. 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.

  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. 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

  8. 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.

  9. Adenylate cyclase and G-proteins as a signal transfer system in the guinea pig inner ear.

    PubMed

    Koch, T; Zenner, H P

    1988-01-01

    In many eukaryotic cells G-proteins play a key role in signal transduction through outer cell membranes. To study this pathway in the auditory organ of mammals we examined tissue preparations from the stria vascularis and the organ of Corti from the guinea pig inner ear. The activity of adenylate cyclase was measured by stimulation at the site of the enzyme, the hormone receptors and the modulating G-proteins. In the organ of Corti we found a low enzyme activity in all cochlear turns. The stria vascularis, however, showed a constant high concentration of beta 2-adrenergic receptors and of stimulating G-proteins in all cochlear turns. In contrast, the activity of the enzyme increased from the apical to the basal turn. Adenylate cyclase could be stimulated or inhibited in a concentration-dependent manner by drugs selectively effecting the G-proteins. Our results suggest a structure of the adenylate cyclase complex in the inner ear similar to other organs. Pathophysiological correlations to hearing loss associated with pseudohypoparathyroidism are discussed.

  10. 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.

  11. Adenylate cyclase and carbonic anhydrase in the semicircular canal epithelium of the frog Rana esculenta. An ultrastructural cytochemical localization.

    PubMed

    Oudar, O; Ferrary, E; Feldmann, G

    1990-12-01

    Because the secretion of endolymph has been localized in the ampullar part of the frog semicircular canal, we attempted to determine by cytochemical methods the ultrastructural localization of two enzymes that are assumed to play a role in endolymph secretion: carbonic anhydrase and adenylate cyclase. Functionally, the epithelium of the frog semicircular canal can be schematically divided into three areas: sensory (crista ampullaris), secretory (dark cells), and non-sensory and nonsecretory (transitional and undifferentiated cells) areas. Carbonic anhydrase activity was widely distributed in dark cells. Dark cell labeling disappeared in the presence of acetazolamide. The other cells of the canal did not show any carbonic anhydrase labeling except for the supporting cells of the sensory cells. Adenylate cyclase activity was found on the basolateral and apical membranes of dark cells, and on the apical membrane of sensory cells; weak labeling was also observed in the other epithelial cells. In the apical membrane of the dark cells, adenylate cyclase labeling was dependent on the presence of vasotocin, the frog antidiuretic hormone. The dark cells of the frog semicircular canal thus possess the enzyme equipment needed for the secretion of endolymph and its possible hormonal regulation.

  12. A Role for Calcium-Activated Adenylate Cyclase and Protein Kinase A in the Lens Src Family Kinase and Na,K-ATPase Response to Hyposmotic Stress.

    PubMed

    Shahidullah, Mohammad; Mandal, Amritlal; Delamere, Nicholas A

    2017-09-01

    Na,K-ATPase activity in lens epithelium is subject to control by Src family tyrosine kinases (SFKs). Previously we showed hyposmotic solution causes an SFK-dependent increase in Na,K-ATPase activity in the epithelium. Here we explored the role of cAMP in the signaling mechanism responsible for the SFK and Na,K-ATPase response. Intact porcine lenses were exposed to hyposmotic Krebs solution (200 mOsm) then the epithelium was assayed for cAMP, SFK phosphorylation (activation) or Na,K-ATPase activity. An increase of cAMP was observed in the epithelium of lenses exposed to hyposmotic solution. In lenses exposed to hyposmotic solution SFK phosphorylation in the epithelium approximately doubled as did Na,K-ATPase activity and both responses were prevented by H89, a protein kinase A inhibitor. The magnitude of the SFK response to hyposmotic solution was reduced by a TRPV4 antagonist HC067047 added to prevent TRPV4-mediated calcium entry, and by a cytoplasmic Ca2+ chelator BAPTA-AM. The Na,K-ATPase activity response in the epithelium of lenses exposed to hyposmotic solution was abolished by BAPTA-AM. As a direct test of cAMP-dependent SFK activation, intact lenses were exposed to 8-pCPT-cAMP, a cell-permeable cAMP analog. 8-pCPT-cAMP caused robust SFK activation. Using Western blot, two calcium-activated adenylyl cyclases, ADCY3 and ADCY8, were detected in lens epithelium. Calcium-activated adenylyl cyclases are expressed in the lens epithelium and SFK activation is linked to a rise of cAMP that occurs upon hyposmotic challenge. The findings point to cAMP as a link between TRPV4 channel-mediated calcium entry, SFK activation, and a subsequent increase of Na,K-ATPase activity.

  13. Microinfusion of pituitary adenylate cyclase-activating polypeptide into the central nucleus of amygdala of the rat produces a shift from an active to passive mode of coping in the shock-probe fear/defensive burying test.

    PubMed

    Legradi, Gabor; Das, Mahasweta; Giunta, Brian; Hirani, Khemraj; Mitchell, E Alice; Diamond, David M

    2007-01-01

    High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) nerve fibers are present in the central nucleus of amygdala (CeA), a brain region implicated in the control of fear-related behavior. This study evaluated PACAPergic modulation of fear responses at the CeA in male Sprague-Dawley rats. PACAP (50-100 pmol) microinfusion via intra-CeA cannulae produced increases in immobility and time the rats spent withdrawn into a corner opposite to the electrified probe compared to controls in the shock-probe fear/defensive burying test. Shock-probe burying and exploration, numbers of shocks received, locomotion distance, and velocity were all reduced by intra-CeA PACAP injection. Further, intra-CeA PACAP effects were manifested only when the animals were challenged by shock, as intra-CeA PACAP injections did not cause significant changes in the behaviors of unshocked rats. Thus, intra-CeA administration of PACAP produces a distinct reorganization of stress-coping behaviors from active (burying) to passive modes, such as withdrawal and immobility. These findings are potentially significant toward enhancing our understanding of the involvement of PACAP and the CeA in the neural basis of fear and anxiety.

  14. Microinfusion of Pituitary Adenylate Cyclase-Activating Polypeptide into the Central Nucleus of Amygdala of the Rat Produces a Shift from an Active to Passive Mode of Coping in the Shock-Probe Fear/Defensive Burying Test

    PubMed Central

    Legradi, Gabor; Das, Mahasweta; Giunta, Brian; Hirani, Khemraj; Mitchell, E. Alice; Diamond, David M.

    2007-01-01

    High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) nerve fibers are present in the central nucleus of amygdala (CeA), a brain region implicated in the control of fear-related behavior. This study evaluated PACAPergic modulation of fear responses at the CeA in male Sprague-Dawley rats. PACAP (50–100 pmol) microinfusion via intra-CeA cannulae produced increases in immobility and time the rats spent withdrawn into a corner opposite to the electrified probe compared to controls in the shock-probe fear/defensive burying test. Shock-probe burying and exploration, numbers of shocks received, locomotion distance, and velocity were all reduced by intra-CeA PACAP injection. Further, intra-CeA PACAP effects were manifested only when the animals were challenged by shock, as intra-CeA PACAP injections did not cause significant changes in the behaviors of unshocked rats. Thus, intra-CeA administration of PACAP produces a distinct reorganization of stress-coping behaviors from active (burying) to passive modes, such as withdrawal and immobility. These findings are potentially significant toward enhancing our understanding of the involvement of PACAP and the CeA in the neural basis of fear and anxiety. PMID:17641738

  15. 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.

  16. 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

  17. Design and Synthesis of Fluorescent Acyclic Nucleoside Phosphonates as Potent Inhibitors of Bacterial Adenylate Cyclases.

    PubMed

    Břehová, Petra; Šmídková, Markéta; Skácel, Jan; Dračínský, Martin; Mertlíková-Kaiserová, Helena; Velasquez, Monica P Soto; Watts, Val J; Janeba, Zlatko

    2016-11-21

    Bordetella pertussis adenylate cyclase toxin (ACT) and Bacillus anthracis edema factor (EF) are key virulence factors with adenylate cyclase (AC) activity that substantially contribute to the pathogenesis of whooping cough and anthrax, respectively. There is an urgent need to develop potent and selective inhibitors of bacterial ACs with prospects for the development of potential antibacterial therapeutics and to study their molecular interactions with the target enzymes. Novel fluorescent 5-chloroanthraniloyl-substituted acyclic nucleoside phosphonates (Cl-ANT-ANPs) were designed and synthesized in the form of their diphosphates (Cl-ANT-ANPpp) as competitive ACT and EF inhibitors with sub-micromolar potency (IC50 values: 11-622 nm). Fluorescence experiments indicated that Cl-ANT-ANPpp analogues bind to the ACT active site, and docking studies suggested that the Cl-ANT group interacts with Phe306 and Leu60. Interestingly, the increase in direct fluorescence with Cl-ANT-ANPpp having an ester linker was strictly calmodulin (CaM)-dependent, whereas Cl-ANT-ANPpp analogues with an amide linker, upon binding to ACT, increased the fluorescence even in the absence of CaM. Such a dependence of binding on structural modification could be exploited in the future design of potent inhibitors of bacterial ACs. Furthermore, one Cl-ANT-ANP in the form of a bisamidate prodrug was able to inhibit B. pertussis ACT activity in macrophage cells with IC50 =12 μm. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Pertussis toxin and adenylate cyclase toxin: key virulence factors of Bordetella pertussis and cell biology tools

    PubMed Central

    Carbonetti, Nicholas H

    2010-01-01

    Pertussis toxin and adenylate cyclase toxin are two important virulence factors of Bordetella pertussis, the bacterial cause of the respiratory disease pertussis or whooping cough. In addition to studies on the structure, function and role in pathogenesis of these two toxins, they are both used as cell biology tools for a variety of applications owing to their ability to enter mammalian cells, perform enzymatic activities and modify cell signaling events. In this article, recent data from the research literature that enhance our understanding of the nature of these two toxins, their role in the pathogenesis of B. pertussis infection and disease, particularly in modulating host immune responses, and their use as tools for other areas of research will be outlined. PMID:20210554

  19. 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.

  20. 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

  1. 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

  2. The Bordetella Adenylate Cyclase Repeat-in-Toxin (RTX) Domain Is Immunodominant and Elicits Neutralizing Antibodies*

    PubMed Central

    Wang, Xianzhe; Maynard, Jennifer A.

    2015-01-01

    The adenylate cyclase toxin (ACT) is a multifunctional virulence factor secreted by Bordetella species. Upon interaction of its C-terminal hemolysin moiety with the cell surface receptor αMβ2 integrin, the N-terminal cyclase domain translocates into the host cell cytosol where it rapidly generates supraphysiological cAMP concentrations, which inhibit host cell anti-bacterial activities. Although ACT has been shown to induce protective immunity in mice, it is not included in any current acellular pertussis vaccines due to protein stability issues and a poor understanding of its role as a protective antigen. Here, we aimed to determine whether any single domain could recapitulate the antibody responses induced by the holo-toxin and to characterize the dominant neutralizing antibody response. We first immunized mice with ACT and screened antibody phage display libraries for binding to purified ACT. The vast majority of unique antibodies identified bound the C-terminal repeat-in-toxin (RTX) domain. Representative antibodies binding two nonoverlapping, neutralizing epitopes in the RTX domain prevented ACT association with J774A.1 macrophages and soluble αMβ2 integrin, suggesting that these antibodies inhibit the ACT-receptor interaction. Sera from mice immunized with the RTX domain showed similar neutralizing activity as ACT-immunized mice, indicating that this domain induced an antibody response similar to that induced by ACT. These data demonstrate that RTX can elicit neutralizing antibodies and suggest it may present an alternative to ACT. PMID:25505186

  3. 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.

  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. 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.

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

    PubMed Central

    Ugur, O; Onaran, H O

    1997-01-01

    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

  7. 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

  8. 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.

  9. Neuritogenesis induced by vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide, and peptide histidine methionine in SH-SY5y cells is associated with regulated expression of cytoskeleton mRNAs and proteins.

    PubMed

    Héraud, Céline; Hilairet, Sandrine; Muller, Jean-Marc; Leterrier, Jean-François; Chadéneau, Corinne

    2004-02-01

    Vasoactive intestinal peptide (VIP) and the related peptides pituitary adenylate cyclase-activating polypeptide (PACAP) and peptide histidine methionine (PHM) are known to regulate proliferation and/or differentiation in normal and tumoral cells. In this study, neuritogenesis in human neuroblastoma SH-SY5Y cells cultured in serum-free medium was induced by VIP, PACAP, and PHM. The establishment of this process was followed by the quantification of neurite length and branching and the expression of neurofilament mRNAs, neurofilament proteins, and other cytoskeletal protein markers of neuronal differentiation: neuron-specific MAPs and beta-tubulin III. Neurite length and branching and the expression of most markers tested were increased by VIP and PACAP in a similar, although slightly different, fashion. In contrast, neuritic elongation induced by PHM was correlated with neither an increase in branching or neurofilament mRNAs nor a clear change in the expression of cytoskeleton proteins, with the exception of the stimulation by PHM of doublecortin, a microtubule-associated marker of migrating neuroblasts. These findings are the first evidence from a human neuron-like cell line for 1) a direct regulation of the metabolism of neurofilaments by VIP and PACAP and 2) the induction by PHM of neuritic processes of an apparent immature character. Copyright 2003 Wiley-Liss, Inc.

  10. 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

  11. 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.

  12. 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

  13. 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

  14. Molecular mechanism of photoactivation of a light-regulated adenylate cyclase.

    PubMed

    Ohki, Mio; Sato-Tomita, Ayana; Matsunaga, Shigeru; Iseki, Mineo; Tame, Jeremy R H; Shibayama, Naoya; Park, Sam-Yong

    2017-08-08

    The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) detects light through a flavin chromophore within the N-terminal BLUF domain. BLUF domains have been found in a number of different light-activated proteins, but with different relative orientations. The two BLUF domains of OaPAC are found in close contact with each other, forming a coiled coil at their interface. Crystallization does not impede the activity switching of the enzyme, but flash cooling the crystals to cryogenic temperatures prevents the signature spectral changes that occur on photoactivation/deactivation. High-resolution crystallographic analysis of OaPAC in the fully activated state has been achieved by cryocooling the crystals immediately after light exposure. Comparison of the isomorphous light- and dark-state structures shows that the active site undergoes minimal changes, yet enzyme activity may increase up to 50-fold, depending on conditions. The OaPAC models will assist the development of simple, direct means to raise the cyclic AMP levels of living cells by light, and other tools for optogenetics.

  15. 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.

  16. 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

  17. 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.

  18. How adenylate cyclase choreographs the pas de deux of the receptors heteromerization dance.

    PubMed

    Woods, A S; Jackson, S N

    2013-05-15

    Our work suggests that heteromer formation, mainly involves linear motifs (LMs) found in disordered regions of proteins. Local disorder imparts plasticity to LMs. Most molecular recognition of proteins occurs between short linear segments, known as LMs. Interaction of short continuous epitopes is not constrained by sequence and has the advantage of resulting in interactions with micromolar affinities which suit transient, reversible complexes such as receptor heteromers. Electrostatic interactions between epitopes of the G-protein coupled receptors (GPCR) involved, are the key step in driving heteromer formation forward. The first step in heteromerization, involves phosphorylating Ser/Thr in an epitope containing a casein kinase 1/2-consensus site. Our data suggest that dopaminergic neurotransmission, through cAMP-dependent protein kinase A (PKA) slows down heteromerization. The negative charge, acquired by the phosphorylation of a Ser/Thr in a PKA consensus site in the Arg-rich epitope, affects the activity of the receptors involved in heteromerization by causing allosteric conformational changes, due to the repulsive effect generated by the negatively charged phosphate. In addition to modulating heteromerization, it affects the stability of the heteromers' interactions and their binding affinity. So here we have an instance where phosphorylation is not just an on/off switch, instead by weakening the noncovalent bond, heteromerization acts like a rheostat that controls the stability of the heteromer through activation or inhibition of adenylate cyclase by the neurotransmitter Dopamine depending on which Dopamine receptor it docks at. Published by Elsevier Ltd.

  19. Albumin, in the Presence of Calcium, Elicits a Massive Increase in Extracellular Bordetella Adenylate Cyclase Toxin.

    PubMed

    Gonyar, Laura A; Gray, Mary C; Christianson, Gregory J; Mehrad, Borna; Hewlett, Erik L

    2017-06-01

    Pertussis (whooping cough), caused by Bordetella pertussis, is resurging in the United States and worldwide. Adenylate cyclase toxin (ACT) is a critical factor in establishing infection with B. pertussis and acts by specifically inhibiting the response of myeloid leukocytes to the pathogen. We report here that serum components, as discovered during growth in fetal bovine serum (FBS), elicit a robust increase in the amount of ACT, and ≥90% of this ACT is localized to the supernatant, unlike growth without FBS, in which ≥90% is associated with the bacterium. We have found that albumin, in the presence of physiological concentrations of calcium, acts specifically to enhance the amount of ACT and its localization to the supernatant. Respiratory secretions, which contain albumin, promote an increase in amount and localization of active ACT that is comparable to that elicited by serum and albumin. The response to albumin is not mediated through regulation of ACT at the transcriptional level or activation of the Bvg two-component system. As further illustration of the specificity of this phenomenon, serum collected from mice that lack albumin does not stimulate an increase in ACT. These data, demonstrating that albumin and calcium act synergistically in the host environment to increase production and release of ACT, strongly suggest that this phenomenon reflects a novel host-pathogen interaction that is central to infection with B. pertussis and other Bordetella species. Copyright © 2017 American Society for Microbiology.

  20. 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)

  1. The Rhizobium etli cyaC product: characterization of a novel adenylate cyclase class.

    PubMed

    Téllez-Sosa, Juan; Soberón, Nora; Vega-Segura, Alicia; Torres-Márquez, María E; Cevallos, Miguel A

    2002-07-01

    Adenylate cyclases (ACs) catalyze the formation of 3',5'-cyclic AMP (cAMP) from ATP. A novel AC-encoding gene, cyaC, was isolated from Rhizobium etli by phenotypic complementation of an Escherichia coli cya mutant. The functionality of the cyaC gene was corroborated by its ability to restore cAMP accumulation in an E. coli cya mutant. Further, overexpression of a malE::cyaC fusion protein allowed the detection of significant AC activity levels in cell extracts of an E. coli cya mutant. CyaC is unrelated to any known AC or to any other protein exhibiting a currently known function. Thus, CyaC represents the first member of a novel class of ACs (class VI). Hypothetical genes of unknown function similar to cyaC have been identified in the genomes of the related bacterial species Mesorhizobium loti, Sinorhizobium meliloti, and Agrobacterium tumefaciens. The cyaC gene is cotranscribed with a gene similar to ohr of Xanthomonas campestris and is expressed only in the presence of organic hydroperoxides. The physiological performance of an R. etli cyaC mutant was indistinguishable from that of the wild-type parent strain both under free-living conditions and during symbiosis.

  2. The Rhizobium etli cyaC Product: Characterization of a Novel Adenylate Cyclase Class

    PubMed Central

    Téllez-Sosa, Juan; Soberón, Nora; Vega-Segura, Alicia; Torres-Márquez, María E.; Cevallos, Miguel A.

    2002-01-01

    Adenylate cyclases (ACs) catalyze the formation of 3′,5′-cyclic AMP (cAMP) from ATP. A novel AC-encoding gene, cyaC, was isolated from Rhizobium etli by phenotypic complementation of an Escherichia coli cya mutant. The functionality of the cyaC gene was corroborated by its ability to restore cAMP accumulation in an E. coli cya mutant. Further, overexpression of a malE::cyaC fusion protein allowed the detection of significant AC activity levels in cell extracts of an E. coli cya mutant. CyaC is unrelated to any known AC or to any other protein exhibiting a currently known function. Thus, CyaC represents the first member of a novel class of ACs (class VI). Hypothetical genes of unknown function similar to cyaC have been identified in the genomes of the related bacterial species Mesorhizobium loti, Sinorhizobium meliloti, and Agrobacterium tumefaciens. The cyaC gene is cotranscribed with a gene similar to ohr of Xanthomonas campestris and is expressed only in the presence of organic hydroperoxides. The physiological performance of an R. etli cyaC mutant was indistinguishable from that of the wild-type parent strain both under free-living conditions and during symbiosis. PMID:12057950

  3. Inhibition of lipolysis by agents acting via adenylate cyclase in fat cells from infants and adults.

    PubMed

    Marcus, C; Sonnenfeld, T; Karpe, B; Bolme, P; Arner, P

    1989-09-01

    The in vitro lipolytic effect of catecholamines is poor during infancy because of enhanced alpha 2-adrenoceptor activity. The mechanisms behind this were investigated in isolated fat cells obtained from 1- to 4-mo-old infants and from adults. The cells were incubated with agents that inhibit lipolysis through distinct receptors coupled to adenylate cyclase via the inhibitory GTP binding coupling protein, Gi. The sensitivity to the alpha 2-adrenoceptor agonist clonidine was 14 times higher in the infant group as compared to the adults, whereas that to an adenosine analogue was 14 times lower. The sensitivities to prostaglandin E2 and nicotinic acid were similar in both age groups. Preincubation of the adipocytes with pertussis toxin abolished the antilipolytic effects of all agents. The density of alpha 2-adrenoceptor binding sites determined with [3H]yohimbine was increased by about 25% in the infants. In conclusion, the antilipolytic sensitivity of adenosine and alpha 2-adrenoceptors develops separately and may play different roles in the regulation of lipolysis in man. Furthermore, the enhanced alpha 2-adrenoceptor sensitivity during infancy seems at least in part to be due to an increase in the number of receptors.

  4. Bordetella adenylate cyclase toxin is a unique ligand of the integrin complement receptor 3

    PubMed Central

    Osicka, Radim; Osickova, Adriana; Hasan, Shakir; Bumba, Ladislav; Cerny, Jiri; Sebo, Peter

    2015-01-01

    Integrins are heterodimeric cell surface adhesion and signaling receptors that are essential for metazoan existence. Some integrins contain an I-domain that is a major ligand binding site. The ligands preferentially engage the active forms of the integrins and trigger signaling cascades that alter numerous cell functions. Here we found that the adenylate cyclase toxin (CyaA), a key virulence factor of the whooping cough agent Bordetella pertussis, preferentially binds an inactive form of the integrin complement receptor 3 (CR3), using a site outside of its I-domain. CyaA binding did not trigger downstream signaling of CR3 in human monocytes and CyaA-catalyzed elevation of cAMP effectively blocked CR3 signaling initiated by a natural ligand. This unprecedented type of integrin-ligand interaction distinguishes CyaA from all other known ligands of the I-domain-containing integrins and provides a mechanistic insight into the previously observed central role of CyaA in the pathogenesis of B. pertussis. DOI: http://dx.doi.org/10.7554/eLife.10766.001 PMID:26650353

  5. 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.

  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. 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.

  10. 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.

  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. 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.

  14. 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.

  15. 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

  16. Kelch-repeat proteins interacting with the Gα protein Gpa2 bypass adenylate cyclase for direct regulation of protein kinase A in yeast

    PubMed Central

    Peeters, Tom; Louwet, Wendy; Geladé, Ruud; Nauwelaers, David; Thevelein, Johan M.; Versele, Matthias

    2006-01-01

    The cAMP–PKA pathway consists of an extracellular ligand-sensitive G protein-coupled receptor, a G protein signal transmitter, and the effector, adenylate cyclase, of which the product, cAMP, acts as an intracellular second messenger. cAMP activates PKA by dissociating the regulatory subunit from the catalytic subunit. Yeast cells (Saccharomyces cerevisiae) contain a glucose/sucrose-sensitive seven-transmembrane domain receptor, Gpr1, that was proposed to activate adenylate cyclase through the Gα protein Gpa2. Consistently, we show here that adenylate cyclase binds only to active, GTP-bound Gpa2. Two related kelch-repeat proteins, Krh1/Gpb2 and Krh2/Gpb1, are associated with Gpa2 and were suggested to act as Gβ mimics for Gpa2, based on their predicted seven-bladed β-propeller structure. However, we find that although Krh1 associates with both GDP and GTP-bound Gpa2, it displays a preference for GTP-Gpa2. The strong down-regulation of PKA targets by Krh1 and Krh2 does not require Gpa2 but is strictly dependent on both the catalytic and the regulatory subunits of PKA. Krh1 directly interacts with PKA by means of the catalytic subunits, and Krh1/2 stimulate the association between the catalytic and regulatory subunits in vivo. Indeed, both a constitutively active GPA2 allele and deletion of KRH1/2 lower the cAMP requirement of PKA for growth. We propose that active Gpa2 relieves the inhibition imposed by the kelch-repeat proteins on PKA, thereby bypassing adenylate cyclase for direct regulation of PKA. Importantly, we show that Krh1/2 also enhance the association between mouse R and C subunits, suggesting that Krh control of PKA has been evolutionarily conserved. PMID:16924114

  17. Modulation of Pertussis and Adenylate Cyclase Toxins by Sigma Factor RpoE in Bordetella pertussis.

    PubMed

    Barbier, Mariette; Boehm, Dylan T; Sen-Kilic, Emel; Bonnin, Claire; Pinheiro, Theo; Hoffman, Casey; Gray, Mary; Hewlett, Erik; Damron, F Heath

    2017-01-01

    Bordetella pertussis is a human pathogen that can infect the respiratory tract and cause the disease known as whooping cough. B. pertussis uses pertussis toxin (PT) and adenylate cyclase toxin (ACT) to kill and modulate host cells to allow the pathogen to survive and persist. B. pertussis encodes many uncharacterized transcription factors, and very little is known about their functions. RpoE is a sigma factor which, in other bacteria, responds to oxidative, heat, and other environmental stresses. RseA is a negative regulator of RpoE that sequesters the sigma factor to regulate gene expression based on conditions. In B. pertussis, deletion of the rseA gene results in high transcriptional activity of RpoE and large amounts of secretion of ACT. By comparing parental B. pertussis to an rseA gene deletion mutant (PM18), we sought to characterize the roles of RpoE in virulence and determine the regulon of genes controlled by RpoE. Despite high expression of ACT, the rseA mutant strain did not infect the murine airway as efficiently as the parental strain and PM18 was killed more readily when inside phagocytes. RNA sequencing analysis was performed and 263 genes were differentially regulated by RpoE, and surprisingly, the rseA mutant strain where RpoE activity was elevated expressed very little pertussis toxin. Western blots and proteomic analysis corroborated the inverse relationship of PT to ACT expression in the high-RpoE-activity rseA deletion strain. Our data suggest that RpoE can modulate PT and ACT expression indirectly through unidentified mechanisms in response to conditions. Copyright © 2016 American Society for Microbiology.

  18. Modulation of Pertussis and Adenylate Cyclase Toxins by Sigma Factor RpoE in Bordetella pertussis

    PubMed Central

    Barbier, Mariette; Boehm, Dylan T.; Sen-Kilic, Emel; Bonnin, Claire; Pinheiro, Theo; Hoffman, Casey; Gray, Mary; Hewlett, Erik

    2016-01-01

    ABSTRACT Bordetella pertussis is a human pathogen that can infect the respiratory tract and cause the disease known as whooping cough. B. pertussis uses pertussis toxin (PT) and adenylate cyclase toxin (ACT) to kill and modulate host cells to allow the pathogen to survive and persist. B. pertussis encodes many uncharacterized transcription factors, and very little is known about their functions. RpoE is a sigma factor which, in other bacteria, responds to oxidative, heat, and other environmental stresses. RseA is a negative regulator of RpoE that sequesters the sigma factor to regulate gene expression based on conditions. In B. pertussis, deletion of the rseA gene results in high transcriptional activity of RpoE and large amounts of secretion of ACT. By comparing parental B. pertussis to an rseA gene deletion mutant (PM18), we sought to characterize the roles of RpoE in virulence and determine the regulon of genes controlled by RpoE. Despite high expression of ACT, the rseA mutant strain did not infect the murine airway as efficiently as the parental strain and PM18 was killed more readily when inside phagocytes. RNA sequencing analysis was performed and 263 genes were differentially regulated by RpoE, and surprisingly, the rseA mutant strain where RpoE activity was elevated expressed very little pertussis toxin. Western blots and proteomic analysis corroborated the inverse relationship of PT to ACT expression in the high-RpoE-activity rseA deletion strain. Our data suggest that RpoE can modulate PT and ACT expression indirectly through unidentified mechanisms in response to conditions. PMID:27849178

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. 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.

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

    PubMed

    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.

  5. 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

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. Properties of rat anterior pituitary vasopressin receptors: relation to adenylate cyclase and the effect of corticotropin-releasing factor.

    PubMed Central

    Gaillard, R C; Schoenenberg, P; Favrod-Coune, C A; Muller, A F; Marie, J; Bockaert, J; Jard, S

    1984-01-01

    Crude plasma membrane fractions were prepared from female Wistar rat anterior pituitaries. These fractions contained a single population of specific 3H-labeled [8-lysine]vasopressin [( 3H]vasopressin) binding sites with a dissociation of constant (Kd) of 8 +/- 2 X 10(-9) M and maximal binding capacity of 244 +/- 45 fmol/mg of protein. The Kd values for a series of vasopressin structural analogues with selective vasopressor or antidiuretic activities were determined together with the corresponding corticotropin-releasing activities (isolated perfused pituitary cells were used). A good correspondence was found between the two sets of values, suggesting that the detected vasopressin binding sites are the receptors involved in vasopressin-induced corticotropin release. The order of potency of these analogues for the binding to hypophysial receptors was similar to that found for the binding to the receptors involved in the vasopressor response. Corticotropin-releasing factor and angiotensin did not affect vasopressin binding to pituitary membranes. Median eminence extracts inhibited [3H]vasopressin binding with an efficiency very close to that expected from their vasopressin content. Corticotropin-releasing factor activated, and angiotensin inhibited, the adenylate cyclase activity of pituitary membranes. Under the same experimental conditions, vasopressin did not influence adenylate cyclase activity nor did it affect the corticotropin-releasing factor-induced activation. These data support the view that vasopressin is one component of the multifactorial regulation of corticotropin release and that it acts through a cAMP-independent pathway. The potentiation by vasopressin of corticotropin-releasing factor-induced cAMP accumulation in intact cells very likely proceeds through indirect mechanisms, which are not expressed in broken cell preparations. PMID:6326152

  12. Role of adenosine 3',5'-monophosphate and the Ri-receptor Gi-coupled adenylate cyclase inhibitory pathway in the mechanism whereby adrenalectomy increases the adenosine antilipolytic effect in rat fat cells.

    PubMed

    de Mazancourt, P; Lacasa, D; Giot, J; Giudicelli, Y

    1989-03-01

    The aim of this study was to establish the mechanism by which adrenalectomy promotes the antilipolytic effect of the adenosine analog (-)-N6-(R-phenyl-isopropyl)adenosine (R-PIA) in rat fat cells. This action of adrenalectomy was not specific for R-PIA, since it was also observed with nicotinic acid and was prevented by phosphodiesterase inhibitors. In contrast, the inhibitory effect of R-PIA and nicotinic acid toward isoproterenol-stimulated cAMP accumulation was unaltered by adrenalectomy regardless of whether phosphodiesterase inhibitors were present. Whatever the conditions used, however, the cAMP levels in adrenalectomized rat adipocytes were one quarter to one third of those in sham-operated rats and remained below the limit over which variations in cAMP had no more influence in lipolysis. Both total and particulate low Km cAMP phosphodiesterase activities per adipocyte were decreased in adrenalectomized rats, but the stimulatory responses of the particulate enzyme to R-PIA remained unchanged. Pertussis toxin-catalyzed ADP ribosylation studies revealed a marked decrease in the total amount of the alpha-subunits of Go and the adenylate cyclase inhibitory regulatory protein Gi after adrenalectomy. However, the inhibitory dose-response curves of adenylate cyclase to R-PIA, nicotinic acid, GTP, guanylylimidodiphosphate, and guanosine 5'-O-(3-thiotriphosphate) were unaltered by adrenalectomy, indicating that the inhibitory function of Gi is unimpaired by adrenalectomy. Lastly, adrenalectomy resulted in a 60% reduction of the Mn2+-stimulated adenylate cyclase activity/adipocyte, which indicates that adrenalectomy causes a defect in adenylate cyclase catalytic activity. Thus, enhanced antilipolytic effects of R-PIA induced by adrenalectomy do not involve increased function of the adenosine receptor Gi-coupled adenylate cyclase inhibitory pathway, but are related to abnormally low intracellular cAMP levels due to defective adenylate cyclase catalytic activity.

  13. Advent and recent advances in research on the role of pituitary adenylate cyclase-activating polypeptide (PACAP) in the regulation of gonadotropic hormone secretion of female rats.

    PubMed

    Köves, Katalin; Kántor, Orsolya; Lakatos, András; Szabó, Enikő; Kirilly, Eszter; Heinzlmann, Andrea; Szabó, Flóra

    2014-11-01

    PACAP (ADCYAP1) was isolated from ovine hypothalami. PACAP activates three distinct receptor types: G-protein coupled PAC1, VPAC1, and VPAC2 with seven transmembrane domains. Eight splice variants of PAC1 receptor are described. A part of the hypothalamic PACAP is released into the hypophyseal portal circulation. Both hypothalamic and pituitary PACAP are involved in the dynamic control of gonadotropic hormone secretion. In female rats, PACAP in the paraventricular nucleus is upregulated in the morning and pituitary PACAP is upregulated in the late evening of the proestrus stage of the reproductive cycle. PACAP mRNA peak in the hypothalamic PVN precedes the LHRH release into the portal circulation. It is supposed that PACAP peak is evoked by the elevated estrogen on proestrous morning. At the beginning of the so-called critical period of the same day, PACAP level starts to decline allowing LHRH release into the portal circulation, resulting in the LH surge that evokes ovulation. Just before the critical period, icv-administered exogenous PACAP blocks the LH surge and ovulation. The blocking effect of PACAP is mediated through CRF and endogenous opioids. The effect of the pituitary-born PACAP depends on the intracellular cross-talk between PACAP and LHRH.

  14. Delivery of Bordetella pertussis adenylate cyclase toxin to target cells via outer membrane vesicles.

    PubMed

    Donato, Gina M; Goldsmith, Cynthia S; Paddock, Christopher D; Eby, Joshua C; Gray, Mary C; Hewlett, Erik L

    2012-02-17

    Bordetella pertussis adenylate cyclase toxin (ACT) intoxicates cells by producing intracellular cAMP. B. pertussis outer membrane vesicles (OMV) contain ACT on their surface (OMV-ACT), but the properties of OMV-ACT were previously unknown. We found that B. pertussis in the lung from a fatal pertussis case contains OMV, suggesting an involvement in pathogenesis. OMV-ACT and ACT intoxicate cells with and without the toxin's receptor CD11b/CD18. Intoxication by ACT is blocked by antitoxin and anti-CD11b antibodies, but not by cytochalasin-D; in contrast, OMV-ACT is unaffected by either antibody and blocked by cytochalasin-D. Thus OMV-ACT can deliver ACT by processes distinct from those of ACT alone. Copyright © 2012 Federation of European Biochemical Societies. All rights reserved.

  15. Delivery of Bordetella pertussis adenylate cyclase toxin to target cells via outer membrane vesicles

    PubMed Central

    Donato, Gina M.; Goldsmith, Cynthia S.; Paddock, Christopher D.; Eby, Joshua C.; Gray, Mary C.; Hewlett, Erik L.

    2012-01-01

    B.pertussis adenylate cyclase toxin (ACT) intoxicates cells by producing intracellular cAMP. B.pertussis outer membrane vesicles (OMV) contain ACT on their surface (OMV-ACT), but the properties of OMV-ACT were previously unknown. We found that B.pertussis in the lung from a fatal pertussis case contains OMV, suggesting an involvement in pathogenesis. OMV-ACT and ACT intoxicate cells with and without the toxin’s receptor CD11b/CD18. Intoxication by ACT is blocked by antitoxin and anti-CD11b antibodies, but not by cytochalasin-D; in contrast, OMV-ACT is unaffected by either antibody and blocked by cytochalasin-D. Thus OMV-ACT can deliver ACT by processes distinct from those of ACT alone. PMID:22289177

  16. Adenylate cyclase 5 is required for melanophore and male pattern development in the guppy (Poecilia reticulata).

    PubMed

    Kottler, Verena A; Künstner, Axel; Koch, Iris; Flötenmeyer, Matthias; Langenecker, Tobias; Hoffmann, Margarete; Sharma, Eshita; Weigel, Detlef; Dreyer, Christine

    2015-09-01

    Guppies (Poecilia reticulata) are colorful fish that have attracted the attention of pigmentation researchers for almost a century. Here, we report that the blond phenotype of the guppy is caused by a spontaneous mutation in the guppy ortholog of adenylate cyclase 5 (adcy5). Using double digest restriction site-associated DNA sequencing (ddRADseq) and quantitative trait locus (QTL) mapping, we linked the blond phenotype to a candidate region of 118 kb, in which we subsequently identified a 2-bp deletion in adcy5 that alters splicing and leads to a premature stop codon. We show that adcy5, which affects life span and melanoma growth in mouse, is required for melanophore development and formation of male orange pigmentation traits in the guppy. We find that some components of the male orange pattern are particularly sensitive to loss of Adcy5 function. Our work thus reveals a function for Adcy5 in patterning of fish color ornaments.

  17. Adenylate Cyclase 6 Determines cAMP Formation and Aquaporin-2 Phosphorylation and Trafficking in Inner Medulla

    PubMed Central

    Tang, Tong; Murray, Fiona; Schroth, Jana; Insel, Paul A.; Fenton, Robert A.; Hammond, H. Kirk

    2010-01-01

    Arginine vasopressin (AVP) enhances water reabsorption in the renal collecting duct by vasopressin V2 receptor (V2R)-mediated activation of adenylyl cyclase (AC), cAMP-promoted phosphorylation of aquaporin-2 (AQP2), and increased abundance of AQP2 on the apical membrane. Multiple isoforms of adenylate cyclase exist, and the roles of individual AC isoforms in water homeostasis are not well understood. Here, we found that levels of AC6 mRNA, the most highly expressed AC isoform in the inner medulla, inversely correlate with fluid intake. Moreover, mice lacking AC6 had lower levels of inner medullary cAMP, reduced abundance of phosphorylated AQP2 (at both serine-256 and serine-269), and lower urine osmolality than wild-type mice. Water deprivation or administration of the V2R agonist dDAVP did not increase urine osmolality of AC6-deficient mice to the levels of wild-type mice. Furthermore, AC6-deficient mice lacked dDAVP-promoted inner medullary cAMP formation and phosphorylation of serine-269 and had attenuated increases in both phosphorylation of serine-256 and apical membrane AQP2 trafficking. In summary, AC6 expression determines inner medullary cAMP formation and AQP2 phosphorylation and trafficking, the absence of which causes nephrogenic diabetes insipidus. PMID:20864687

  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. 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

  20. Interaction of the antiarrhythmic agents SR 33589 and amiodarone with the beta-adrenoceptor and adenylate cyclase in rat heart.

    PubMed Central

    Chatelain, P.; Meysmans, L.; Mattéazzi, J. R.; Beaufort, P.; Clinet, M.

    1995-01-01

    1. The effects of SR 33589 and amiodarone on the cardiac beta-adrenoceptor were studied in vitro and after chronic treatment by means of [125I]-(-)-iodocyanopindolol ([125I]-(-)-CYP) binding and measurement of adenylate cyclase activity. 2. Binding of [125I]-(-)-CYP was inhibited in a dose-dependent manner by SR 33589 (IC50=1.8 +/- 0.4 microM, nH=0.93 +/- 0.06) and amiodarone (IC50=8.7 +/- 2.0 microM, nH=9.2 +/- 0.03). Saturation binding experiments indicated a non-competitive interaction such that SR 33589 (1 and 3 microM) and amiodarone (5 and 10 microM) reduced the Bmax of [125I]-(-)-CYP binding without any effect on the KD. Kinetic studies showed that the rate of association of [125I]-(-)-CYP was unchanged while the rate of dissociation was increased both in the presence of SR 33589 (10 microM) and amiodarone (30 microM).3. Under the same conditions, the receptor stimulated adenylate cyclase activity was inhibited in a dose-dependent, but non-competitive manner, by SR 33589 (isoprenaline-, glucagon- and secretin-stimulated enzyme inhibited 50% at 6.8 +/- 0.6 microM, 31 +/- 10 microM and 12 +/- 3 microM, respectively) while the basal, GTP- and GPP(NH)p-stimulated enzyme was inhibited by 5-10% and the NaF and forskolin-stimulated enzyme by 50% at 500 microM. Amiodarone exhibited a similar pattern of inhibition. 4. After chronic oral treatment (50, 100, 150 mg kg(-1) per day, 14 days), both SR 33589 and amiodarone produced a dose-dependent decrease in Bmax without any effect on KD as determined from [125I]-(-)-CYP saturation experiments and a decrease of the isoprenaline- and glucagon-stimulated adenylate cyclase activity without any effect on basal enzyme activity or activity when stimulated by agents acting directly on regulatory catalytic units. 5. Unlike amiodarone, SR 33589 does not contain iodine substituents. Plasma levels of T3, T4, and rT3 were changed after SR 33589 treatment except a decrease in T4 level at the highest dose whilst the T4 T3 ratio and

  1. High throughput synthetic lethality screen reveals a tumorigenic role of adenylate cyclase in fumarate hydratase-deficient cancer cells

    PubMed Central

    2014-01-01

    Background Synthetic lethality is an appealing technique for selectively targeting cancer cells which have acquired molecular changes that distinguish them from normal cells. High-throughput RNAi-based screens have been successfully used to identify synthetic lethal pathways with well-characterized tumor suppressors and oncogenes. The recent identification of metabolic tumor suppressors suggests that the concept of synthetic lethality can be applied to selectively target cancer metabolism as well. Results Here, we perform a high-throughput RNAi screen to identify synthetic lethal genes with fumarate hydratase (FH), a metabolic tumor suppressor whose loss-of-function has been associated with hereditary leiomyomatosis and renal cell carcinoma (HLRCC). Our unbiased screen identified synthetic lethality between FH and several genes in heme metabolism, in accordance with recent findings. Furthermore, we identified an enrichment of synthetic lethality with adenylate cyclases. The effects were validated in an embryonic kidney cell line (HEK293T) and in HLRCC-patient derived cells (UOK262) via both genetic and pharmacological inhibition. The reliance on adenylate cyclases in FH-deficient cells is consistent with increased cyclic-AMP levels, which may act to regulate cellular energy metabolism. Conclusions The identified synthetic lethality of FH with adenylate cyclases suggests a new potential target for treating HLRCC patients. PMID:24568598

  2. High throughput synthetic lethality screen reveals a tumorigenic role of adenylate cyclase in fumarate hydratase-deficient cancer cells.

    PubMed

    Boettcher, Michael; Lawson, Andrew; Ladenburger, Viola; Fredebohm, Johannes; Wolf, Jonas; Hoheisel, Jörg D; Frezza, Christian; Shlomi, Tomer

    2014-02-25

    Synthetic lethality is an appealing technique for selectively targeting cancer cells which have acquired molecular changes that distinguish them from normal cells. High-throughput RNAi-based screens have been successfully used to identify synthetic lethal pathways with well-characterized tumor suppressors and oncogenes. The recent identification of metabolic tumor suppressors suggests that the concept of synthetic lethality can be applied to selectively target cancer metabolism as well. Here, we perform a high-throughput RNAi screen to identify synthetic lethal genes with fumarate hydratase (FH), a metabolic tumor suppressor whose loss-of-function has been associated with hereditary leiomyomatosis and renal cell carcinoma (HLRCC). Our unbiased screen identified synthetic lethality between FH and several genes in heme metabolism, in accordance with recent findings. Furthermore, we identified an enrichment of synthetic lethality with adenylate cyclases. The effects were validated in an embryonic kidney cell line (HEK293T) and in HLRCC-patient derived cells (UOK262) via both genetic and pharmacological inhibition. The reliance on adenylate cyclases in FH-deficient cells is consistent with increased cyclic-AMP levels, which may act to regulate cellular energy metabolism. The identified synthetic lethality of FH with adenylate cyclases suggests a new potential target for treating HLRCC patients.

  3. 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

  4. PPARγ-Dependent Regulation of Adenylate Cyclase 6 Amplifies the Stimulatory Effect of cAMP on Renin Gene Expression

    PubMed Central

    Desch, Michael; Schubert, Thomas; Schreiber, Andrea; Mayer, Sandra; Friedrich, Björn; Artunc, Ferruh; Todorov, Vladimir T.

    2010-01-01

    The second messenger cAMP plays an important role in the regulation of renin gene expression. Nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) is known to stimulate renin gene transcription acting through PPARγ-binding sequences in renin promoter. We show now that activation of PPARγ by unsaturated fatty acids or thiazolidinediones drastically augments the cAMP-dependent increase of renin mRNA in the human renin-producing cell line Calu-6. The underlying mechanism involves potentiation of agonist-induced cAMP increase and up-regulation of adenylate cyclase 6 (AC6) gene expression. We identified a palindromic element with a 3-bp spacer (Pal3) in AC6 intron 1 (AC6Pal3). AC6Pal3 bound PPARγ and mediated trans-activation by PPARγ agonist. AC6 knockdown decreased basal renin mRNA level and attenuated the maximal PPARγ-dependent stimulation of the cAMP-induced renin gene expression. AC6Pal3 decoy oligonucleotide abrogated the PPARγ-dependent potentiation of cAMP-induced renin gene expression. Treatment of mice with PPARγ agonist increased AC6 mRNA kidney levels. Our data suggest that in addition to its direct effect on renin gene transcription, PPARγ “sensitizes” renin gene to cAMP via trans-activation of AC6 gene. AC6 has been identified as PPARγ target gene with a functional Pal3 sequence. PMID:20861226

  5. 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.

  6. 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.

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

    PubMed

    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.

  9. 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.

  10. 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

  11. [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.

  12. 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.

  13. 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.

  14. 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.

  15. Use of a Toxin Neutralization Assay To Characterize the Serologic Response to Adenylate Cyclase Toxin after Infection with Bordetella pertussis.

    PubMed

    Eby, Joshua C; Gray, Mary C; Warfel, Jason M; Merkel, Tod J; Hewlett, Erik L

    2017-01-01

    Adenylate cyclase toxin (ACT) is an essential virulence factor of Bordetella pertussis, and antibodies to ACT protect against B. pertussis infection in mice. The toxin is therefore a strong candidate antigen for addition to future acellular pertussis vaccines. In order to characterize the functionality of the immunologic response to ACT after infection, we developed an assay for testing the ability of serum samples from subjects infected with B. pertussis to neutralize ACT-induced cytotoxicity in J774 macrophage cells. Baboons develop neutralizing anti-ACT antibodies following infection with B. pertussis, and all sera from baboons with positive anti-ACT IgG enzyme-linked immunosorbent assay (ELISA) results neutralized ACT cytotoxicity. The toxin neutralization assay (TNA) was positive in some baboon sera in which ELISA remained negative. Of serum samples obtained from humans diagnosed with pertussis by PCR, anti-ACT IgG ELISA was positive in 72%, and TNA was positive in 83%. All samples positive for anti-ACT IgG ELISA were positive by TNA, and none of the samples from humans without pertussis neutralized toxin activity. These findings indicate that antibodies to ACT generated following infection with B. pertussis consistently neutralize toxin-induced cytotoxicity and that TNA can be used to improve understanding of the immunologic response to ACT after infection or vaccination. Copyright © 2017 American Society for Microbiology.

  16. 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.

  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. 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

  19. 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.

  20. 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.

  1. Adenylate cyclase orthologues in two filamentous entomopathogens contribute differentially to growth, conidiation, pathogenicity, and multistress responses.

    PubMed

    Wang, Jie; Zhou, Gang; Ying, Sheng-Hua; Feng, Ming-Guang

    2014-04-01

    Adenylate cyclase (AC) is a core element of cAMP signalling network. Here we show functional diversity and differentiation of Beauveria bassiana AC (BbAC) and Metarhizium robertsii AC (MrAC). Severe growth defects occurred in ΔBbAC and ΔMrAC grown on nutrition-rich SDAY and several minimal media but were largely alleviated by adding cAMP to SDAY. Conidial yield increased greatly in ΔBbAC but decreased in ΔMrAC. During colony growth, ΔBbAC was highly sensitive to oxidation, high osmolarity, cell wall perturbation, carbendazim fungicide, Mn(2+), Zn(2+), Fe(3+), and EDTA but more tolerant to Cu(2+) while ΔMrAC showed higher osmotolerance, decreased sensitivity to Fe(3+), and null response to carbendazim or cell wall stress despite similar responses to oxidation and other metal ions. Conidial UV-B resistance decreased by 32% in ΔBbAC and 22% in ΔMrAC despite little change in their theromotolerance. Median lethal time (LT50) estimates of ΔBbAC and ΔMrAC against susceptible insects were 10.9 and 1.4 d longer than those from wild-type strains respectively. All the phenotypic changes were restored to wild-type levels by each gene complementation. Taken together, BbAC and MrAC regulated differentially conidiation, pathogenicity, and multistress responses in B. bassiana and M. robertsii, thereby making different contributions to their biocontrol potential. Copyright © 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  2. Quantification of the adenylate cyclase toxin of Bordetella pertussis in vitro and during respiratory infection.

    PubMed

    Eby, Joshua C; Gray, Mary C; Warfel, Jason M; Paddock, Christopher D; Jones, Tara F; Day, Shandra R; Bowden, James; Poulter, Melinda D; Donato, Gina M; Merkel, Tod J; Hewlett, Erik L

    2013-05-01

    Whooping cough results from infection of the respiratory tract with Bordetella pertussis, and the secreted adenylate cyclase toxin (ACT) is essential for the bacterium to establish infection. Despite extensive study of the mechanism of ACT cytotoxicity and its effects over a range of concentrations in vitro, ACT has not been observed or quantified in vivo, and thus the concentration of ACT at the site of infection is unknown. The recently developed baboon model of infection mimics the prolonged cough and transmissibility of pertussis, and we hypothesized that measurement of ACT in nasopharyngeal washes (NPW) from baboons, combined with human and in vitro data, would provide an estimate of the ACT concentration in the airway during infection. NPW contained up to ≈ 10(8) CFU/ml B. pertussis and 1 to 5 ng/ml ACT at the peak of infection. Nasal aspirate specimens from two human infants with pertussis contained bacterial concentrations similar to those in the baboons, with 12 to 20 ng/ml ACT. When ≈ 10(8) CFU/ml of a laboratory strain of B. pertussis was cultured in vitro, ACT production was detected in 60 min and reached a plateau of ≈ 60 ng/ml in 6 h. Furthermore, when bacteria were brought into close proximity to target cells by centrifugation, intoxication was increased 4-fold. Collectively, these data suggest that at the bacterium-target cell interface during infection of the respiratory tract, the concentration of ACT can exceed 100 ng/ml, providing a reference point for future studies of ACT and pertussis pathogenesis.

  3. Quantification of the Adenylate Cyclase Toxin of Bordetella pertussis In Vitro and during Respiratory Infection

    PubMed Central

    Eby, Joshua C.; Gray, Mary C.; Warfel, Jason M.; Paddock, Christopher D.; Jones, Tara F.; Day, Shandra R.; Bowden, James; Poulter, Melinda D.; Donato, Gina M.; Merkel, Tod J.

    2013-01-01

    Whooping cough results from infection of the respiratory tract with Bordetella pertussis, and the secreted adenylate cyclase toxin (ACT) is essential for the bacterium to establish infection. Despite extensive study of the mechanism of ACT cytotoxicity and its effects over a range of concentrations in vitro, ACT has not been observed or quantified in vivo, and thus the concentration of ACT at the site of infection is unknown. The recently developed baboon model of infection mimics the prolonged cough and transmissibility of pertussis, and we hypothesized that measurement of ACT in nasopharyngeal washes (NPW) from baboons, combined with human and in vitro data, would provide an estimate of the ACT concentration in the airway during infection. NPW contained up to ∼108 CFU/ml B. pertussis and 1 to 5 ng/ml ACT at the peak of infection. Nasal aspirate specimens from two human infants with pertussis contained bacterial concentrations similar to those in the baboons, with 12 to 20 ng/ml ACT. When ∼108 CFU/ml of a laboratory strain of B. pertussis was cultured in vitro, ACT production was detected in 60 min and reached a plateau of ∼60 ng/ml in 6 h. Furthermore, when bacteria were brought into close proximity to target cells by centrifugation, intoxication was increased 4-fold. Collectively, these data suggest that at the bacterium-target cell interface during infection of the respiratory tract, the concentration of ACT can exceed 100 ng/ml, providing a reference point for future studies of ACT and pertussis pathogenesis. PMID:23429530

  4. Membrane restructuring by Bordetella pertussis adenylate cyclase toxin, a member of the RTX toxin family.

    PubMed

    Martín, César; Requero, M-Asunción; Masin, Jiri; Konopasek, Ivo; Goñi, Félix M; Sebo, Peter; Ostolaza, Helena

    2004-06-01

    Adenylate cyclase toxin (ACT) is secreted by Bordetella pertussis, the bacterium causing whooping cough. ACT is a member of the RTX (repeats in toxin) family of toxins, and like other members in the family, it may bind cell membranes and cause disruption of the permeability barrier, leading to efflux of cell contents. The present paper summarizes studies performed on cell and model membranes with the aim of understanding the mechanism of toxin insertion and membrane restructuring leading to release of contents. ACT does not necessarily require a protein receptor to bind the membrane bilayer, and this may explain its broad range of host cell types. In fact, red blood cells and liposomes (large unilamellar vesicles) display similar sensitivities to ACT. A varying liposomal bilayer composition leads to significant changes in ACT-induced membrane lysis, measured as efflux of fluorescent vesicle contents. Phosphatidylethanolamine (PE), a lipid that favors formation of nonlamellar (inverted hexagonal) phases, stimulated ACT-promoted efflux. Conversely, lysophosphatidylcholine, a micelle-forming lipid that opposes the formation of inverted nonlamellar phases, inhibited ACT-induced efflux in a dose-dependent manner and neutralized the stimulatory effect of PE. These results strongly suggest that ACT-induced efflux is mediated by transient inverted nonlamellar lipid structures. Cholesterol, a lipid that favors inverted nonlamellar phase formation and also increases the static order of phospholipid hydrocarbon chains, among other effects, also enhanced ACT-induced liposomal efflux. Moreover, the use of a recently developed fluorescence assay technique allowed the detection of trans-bilayer (flip-flop) lipid motion simultaneous with efflux. Lipid flip-flop further confirms the formation of transient nonlamellar lipid structures as a result of ACT insertion in bilayers.

  5. 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.

  6. 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.

  7. 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.

  8. Effect of cardiopulmonary bypass on beta adrenergic receptor-adenylate cyclase system on surfaces of peripheral lymphocytes.

    PubMed

    Luo, A; Tian, Y; Jin, S

    2000-01-01

    The experimental results showed that the level of CAMP, the ratio of cAPM to cGMP, IL-2R expression and IL-2 production in vitro in lymphocytes immediate and 2 weeks after cardiopulmonary bypass (CPB) were significantly lower than those before anesthetics in the patients undergoing cardiac surgery with CPB. These findings suggested that CPB could cause serious damage to adrenergic beta receptor-adenylate cyclase system on circulating lymphocytes surfaces, which might be one of the mechanisms resulting in immunosuppression after open heart surgery with CPB.

  9. Region-Specific Disruption of Adenylate Cyclase Type 1 Gene Differentially Affects Somatosensorimotor Behaviors in Mice1,2,3

    PubMed Central

    Arakawa, Hiroyuki; Akkentli, Fatih

    2014-01-01

    Abstract Cover Figure Region-specific adenylyl cyclase 1 (AC1) loss of function differentially affects both patterning and sensorimotor behaviors in mice. AC1 is expressed at all levels of the somatosensory pathway and plays a major role in refinement and patterning of topographic sensory maps. Cortex-specific AC1 loss of function (CxAC1KO mice) does not affect barrel patterning and activation of specific barrels corresponding to stimulated whiskers and does not impair sensorimotor behaviors. While global (AC1KO) and thalamus-specific (ThAC1KO) AC1 loss of function leads to absence of barrel patterns, selective whisker stimulation activates topographically aligned cortical loci. Despite functional topography of the whisker-barrel cortex, sensorimotor and social behaviors are impaired, indicating the importance of patterning of topographical sensory maps in the neocortex. Adenylate cyclase type I (AC1) is primarily, and, abundantly, expressed in the brain. Intracellular calcium/calmodulin increases regulate AC1 in an activity-dependent manner. Upon stimulation, AC1 produces cAMP and it is involved in the patterning and the refinement of neural circuits. In mice, spontaneous mutations or targeted deletion of the Adcy1 gene, which encodes AC1, resulted in neuronal pattern formation defects. Neural modules in the primary somatosensory (SI) cortex, the barrels, which represent the topographic distribution of the whiskers on the snout, failed to form (Welker et al., 1996; Abdel-Majid et al., 1998). Cortex- or thalamus-specific Adcy1 deletions led to different cortical pattern phenotypes, with thalamus-specific disruption phenotype being more severe (Iwasato et al., 2008; Suzuki et al., 2013). Despite the absence of barrels in the “barrelless”/Adcy1 null mice, thalamocortical terminal bouton density and activation of cortical zones following whisker stimulation were roughly topographic (Abdel-Majid et al., 1998; Gheorghita et al., 2006). To what extent does patterning

  10. Regulation of adenylate cyclase synthesis in Escherichia coli: studies with cya-lac operon and protein fusion strains.

    PubMed Central

    Bankaitis, V A; Bassford, P J

    1982-01-01

    We have isolated cya-lac operon and protein fusions in Escherichia coli K-12, and we used these to study the regulation of cya, the structural gene for adenylate cyclase. Data obtained from these fusion strains suggest that neither cyclic AMP (cAMP) nor the cAMP receptor protein plays a major role in transcriptional or translational regulation of cya expression. Modulation of intracellular cAMP concentrations elicited only weak repression of cya-lac fusion activity under conditions of high intracellular cAMP, relative to fusion activity under conditions of low intracellular cAMP. The functional cAMP receptor protein was required for this effect. Incorporation of delta crp into cya-lac fusion strains did not affect fusion expression in glucose-grown cells as compared with similarly cultured isogenic crp+ strains. Furthermore, 20 independently obtained mutants derived from a cya-lacZ protein fusion strain exhibiting a weak Lac+ phenotype were isolated, and it was determined that the mutants had beta-galactosidase activities ranging from 2- to 77-fold greater than those of the parental strain. None of the mutations responsible for this increase in fusion activity map in the crp locus. We used these mutants to aid in the identification of a 160,000-dalton cya-lacZ hybrid protein. Finally, chromosome mobilization experiments, using cya-lac fusion strains, allowed us to infer a clockwise direction of transcription for the cya gene relative to the standard E. coli genetic map. Images PMID:6286596

  11. Hepatic adenylate cyclase 3 is upregulated by Liraglutide and subsequently plays a protective role in insulin resistance and obesity.

    PubMed

    Liang, Y; Li, Z; Liang, S; Li, Y; Yang, L; Lu, M; Gu, H F; Xia, N

    2016-01-25

    Recent studies have demonstrated that adenylate cyclase 3 (AC3) has a protective role in obesity. This gene resides at the pathway with glucagon-like peptide (GLP)-1. Liraglutide is a GLP-1 analog and has independent glucose and body weight (BW)-reducing effects. In the present study, we aimed to examine whether hepatic AC3 activity was regulated by Liraglutide and to further understand the effect of AC3 in reduction of BW and insulin resistance. The diabesity and obese mice were induced from db/db and C57BL/6 J mice, respectively, by high-fat diet. Liraglutide (0.1 mg kg(-1) per 12 h) was given to the mice twice daily for 12 weeks. C57BL/6 J mice fed with chow diet and obese or diabesity mice treated with saline were used as the controls. Hepatic AC3 gene expression at mRNA and protein levels was analyzed with real-time reverse transcription-PCR and western blot. Fasting blood glucose and serum insulin levels were measured and followed insulin resistance index (HOMA-IR) was evaluated according to the homeostasis model assessment. After administration of Liraglutide, BW and HOMA-IR in obese and diabesity mice were decreased, whereas hepatic AC3 mRNA and protein expression levels were upregulated. The AC3 gene expression was negatively correlated with BW, HOMA-IR and the area ratio of hepatic fat deposition in the liver. The present study thus provides the evidence that hepatic AC3 gene expression is upregulated by Liraglutide. The reduction of BW and improvement of insulin resistance with Liraglutide may be partially explained by AC3 activation.

  12. 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

  13. 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.

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

    PubMed Central

    Duvall, Laura B.; Taghert, Paul H.

    2012-01-01

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

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

    PubMed

    Duvall, Laura B; Taghert, Paul H

    2012-01-01

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

  16. 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

  17. Alternating Hemiplegia of Childhood as a New Presentation of Adenylate Cyclase 5-Mutation-Associated Disease: A Report of Two Cases.

    PubMed

    Westenberger, Ana; Max, Christoph; Brüggemann, Norbert; Domingo, Aloysius; Grütz, Karen; Pawlack, Heike; Weissbach, Anne; Kühn, Andrea A; Spiegler, Juliane; Lang, Anthony E; Sperner, Jürgen; Fung, Victor S C; Schallner, Jens; Gillessen-Kaesbach, Gabriele; Münchau, Alexander; Klein, Christine

    2017-02-01

    Mutations in the adenylate cyclase 5 (ADCY5) gene recently have been identified as the cause of a childhood-onset disorder characterized by persistent or paroxysmal choreic, myoclonic, and/or dystonic movements. The 2 novel mutations we identified expand the clinical spectrum of ADCY5 mutations to include alternating hemiplegia of childhood. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Detection and analysis of agonist-induced formation of the complex of the stimulatory guanine nucleotide-binding protein with adenylate cyclase in intact wild-type and beta 2-adrenoceptor-expressing NG108-15 cells.

    PubMed

    Kim, G D; Carr, I C; Milligan, G

    1995-05-15

    Neuroblastoma x glioma hybrid, NG108-15, cells appear to express the alpha-subunit of the guanine nucleotide-binding protein Gs in a substantial molar excess over its effector adenylate cyclase [Kim, Adie and Milligan (1994) Eur. J. Biochem. 219, 135-143]. Addition of the IP prostanoid receptor agonist iloprost to intact NG108-15 cells resulted in a dose-dependent increase in formation of the complex between Gs alpha and adenylate cyclase (GSAC) as measured by specific high-affinity binding of [3H]forskolin. NG108-15 cells transfected to express either relatively high (clone beta N22) or low (clone beta N17) levels of beta 2-adrenoceptor both showed dose-dependent increases in specific [3H]forskolin binding in response to the beta-adrenoceptor agonist isoprenaline, and maximally effective concentrations of isoprenaline resulted in the generation of similar numbers of GSAC complexes in both clones. The dose-effect curve for clone beta N22, however, was some 15-fold to the left of that for clone beta N17, which is similar to that noted for isoprenaline-mediated stimulation of adenylate cyclase activity [Adie and Milligan (1994) Biochem. J. 303, 803-808]. In contrast, dose-effect curves for iloprost stimulation of [3H]forskolin binding were not different in clones beta N22 and beta N17. Basal specific [3H]forskolin binding in the absence of agonist was significantly greater in cells of clone beta N22 than clone beta N17. This was not a reflection of higher immunological levels of adenylate cyclase, indicating that the higher basal formation of GSAC probably reflects empty-receptor activation of Gs. This higher basal specific [3H]forskolin binding was partially reversed by propranolol. The addition of the opioid peptide D-Ala-D-Leu-enkephalin to NG108-15 cells did not reduce iloprost-stimulated [3H]forskolin binding even though this peptide inhibits stimulated adenylate cyclase activity by activation of a delta opioid receptor.

  19. Chemosignal transduction in the vomeronasal organ of garter snakes: cloning of a gene encoding adenylate cyclase from the vomeronasal organ of garter snakes.

    PubMed

    Liu, W; Wang, D; Liu, J; Chen, P; Halpern, M

    1998-10-15

    We previously reported that ES20-receptor binding activates phosphoinositide (PI) turnover, resulting in an increase in inositol-1,4,5-trisphosphate, which in turn mobilizes intracellularly stored calcium in the vomeronasal (VN) sensory epithelium of garter snakes. We also found that the activity of adenylate cyclase (AC) in the VN organ is very sensitive to Ca2+ but insensitive to calmodulin regulation. A 250-bp fragment of adenylate cyclase type VI (AC-VI) was obtained from brain cDNA of garter snake by RT-PCR with degenerate primers. The 250-bp fragments were amplified, cloned, and sequenced. Both Northern blot and RNase protection assays revealed that the vomeronasal organ (VNO) and brain contained more abundance of AC type VI than the main olfactory epithelium. A 3.8-kb cDNA was then cloned from the vomeronasal cDNA library of garter snakes and sequenced. The 5' cDNA was obtained by means of 5' RACE PCR and sequenced. We have successfully cloned a 5200-nucleotide cDNA from VNO of garter snakes containing an open reading frame++ encoding 1150 amino acids of AC-VI protein. The vomeronasal AC is termed AC(VN) . AC(VN) shows a high degree of homology with type VI AC of rat, mouse, or human. In situ hybridization with digoxigenin-labeled cRNA demonstrated that AC(VN) mRNA was abundant in the sensory epithelium but not in the nonsensory epithelium of the mushroom body of the vomeronasal organ of garter snakes. Copyright 1998 Academic Press.

  20. Characterization of Post-Translational Modifications and Cytotoxic Properties of the Adenylate-Cyclase Hemolysin Produced by Various Bordetella pertussis and Bordetella parapertussis Isolates.

    PubMed

    Bouchez, Valérie; Douché, Thibaut; Dazas, Mélody; Delaplane, Sophie; Matondo, Mariette; Chamot-Rooke, Julia; Guiso, Nicole

    2017-09-26

    Bordetella pertussis and Bordetella parapertussis are the causal agents of whooping cough in humans. They produce diverse virulence factors, including adenylate cyclase-hemolysin (AC-Hly), a secreted toxin of the repeat in toxins (RTX) family with cyclase, pore-forming, and hemolytic activities. Post-translational modifications (PTMs) are essential for the biological activities of the toxin produced by B. pertussis. In this study, we compared AC-Hly toxins from various clinical isolates of B. pertussis and B. parapertussis, focusing on (i) the genomic sequences of cyaA genes, (ii) the PTMs of partially purified AC-Hly, and (iii) the cytotoxic activity of the various AC-Hly toxins. The genes encoding the AC-Hly toxins of B. pertussis and B. parapertussis displayed very limited polymorphism in each species. Most of the sequence differences between the two species were found in the C-terminal part of the protein. Both toxins harbored PTMs, mostly corresponding to palmitoylations of the lysine 860 residue and palmoylations and myristoylations of lysine 983 for B. pertussis and AC-Hly and palmitoylations of lysine 894 and myristoylations of lysine 1017 for B. parapertussis AC-Hly. Purified AC-Hly from B. pertussis was cytotoxic to macrophages, whereas that from B. parapertussis was not.

  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. Cyclic AMP-Mediated Suppression of Neutrophil Extracellular Trap Formation and Apoptosis by the Bordetella pertussis Adenylate Cyclase Toxin

    PubMed Central

    Gray, Mary C.; Hewlett, Erik L.

    2014-01-01

    The adenylate cyclase toxin (ACT) of Bordetella pertussis intoxicates target cells by generating supraphysiologic levels of intracellular cyclic AMP (cAMP). Since ACT kills macrophages rapidly and potently, we asked whether ACT would also kill neutrophils. In fact, ACT prolongs the neutrophil life span by inhibiting constitutive apoptosis and preventing apoptosis induced by exposure to live B. pertussis. Imaging of B. pertussis-exposed neutrophils revealed that B. pertussis lacking ACT induces formation of neutrophil extracellular traps (NETs), whereas wild-type B. pertussis does not, suggesting that ACT suppresses NET formation. Indeed, ACT inhibits formation of NETs by generating cAMP and consequently inhibiting the oxidative burst. Convalescent-phase serum from humans following clinical pertussis blocks the ACT-mediated suppression of NET formation. These studies provide novel insight into the phagocyte impotence caused by ACT, which not only impairs neutrophil function but also inhibits death of neutrophils by apoptosis and NETosis. PMID:25287922

  3. Systematic analysis of gene expression alterations and clinical outcomes of adenylate cyclase-associated protein in cancer

    PubMed Central

    Xie, Shuanshuan; Shen, Changxing; Tan, Min; Li, Ming; Song, Xiaolian; Wang, Changhui

    2017-01-01

    Adenylate Cyclase-associated protein (CAP) is an evolutionarily conserved protein that regulates actin dynamics. Our previous study indicates that CAP1 is overexpressed in NSCLC tissues and correlated with poor clinical outcomes, but CAP1 in HeLa cells actually inhibited migration and invasion, the role of CAP was discrepancy in different cancer types. The present study aims to determine whether CAP can serve as a prognostic marker in human cancers. The CAP expression was assessed using Oncomine database to determine the gene alteration during carcinogenesis, the copy number alteration, or mutations of CAP using cBioPortal, International Cancer Genome Consortium, and Tumorscape database investigated, and the association between CAP expression and the survival of cancer patient using Kaplan-Meier plotter and PrognoScan database evaluated. Therefore, the functional correlation between CAP expression and cancer phenotypes can be established; wherein CAP might serve as a diagnostic marker or therapeutic target for certain types of cancers. PMID:28423713

  4. 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

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

    USDA-ARS?s Scientific Manuscript database

    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...

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. The complete amino acid sequence of ubiquitin, an adenylate cyclase stimulating polypeptide probably universal in living cells.

    PubMed

    Schlesinger, D H; Goldstein, G; Niall, H D

    1975-05-20

    The complete amino acid sequence was determined for bovine ubiquitin, and adenylate cyclase stimulating polypeptide, which is probably represented universally in living cells. Ubiquitin has a molecular weight of 8451 and consists of a single polypeptide chain containing 74 amino acid residues. It contains four arginine residues but no cysteine or trytophan residues. The first 61 amino acid residues were obtained by automated Edman degradations. Tryptic digestion of maleated ubiquitin yielded four peptide fragments that were resolved by molecular sieve chromatography and coded in order of decreasing chain length (MT-1, MT-2, MT-3, and MT-4). The automated sequenator determinations on native ubiquintin provided overlapping sequence data for three of these fragments that gave an order of MT-1, MT-3, and then MT-2; Peptide MT-4, a dipeptide, was therefore assigned to the C terminus, and the placement of peptide MT-2 was corroborated by analysis of data from carboxypeptidase digestions of maleated ubiquitin. Peptide MT-2 was domaleated and sequenced by manual Edman degradations through a single lysine residue. It was cleaved at this residue with trypsin, and the two resultant peptides were separated by ion-exchange chromatography. Manual sequencing of the C-terminal demaleated tryptic peptide of MT-2 completed the sequence of MT-2 and that of native ubiquitin. The sequence of ubiquitin was further confirmed and supported by amino acid and parital sequence anlysis of fragments obtained by digestion of maleated ubiquitin with chymotrypsin or staphylococcal protease.

  12. 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.

  13. Expression of novel neurotrophin-1/B-cell stimulating factor-3 (NNT-1/BSF-3) in murine pituitary folliculostellate TtT/GF cells: pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal peptide-induced stimulation of NNT-1/BSF-3 is mediated by protein kinase A, protein kinase C, and extracellular-signal-regulated kinase1/2 pathways.

    PubMed

    Vlotides, George; Zitzmann, Kathrin; Hengge, Sabine; Engelhardt, Dieter; Stalla, Gunter K; Auernhammer, Christoph J

    2004-02-01

    Novel neurotrophin-1/B cell stimulating factor-3 (NNT-1/BSF-3) is a gp130 cytokine potently stimulating corticotroph proopiomelanocortin gene expression and ACTH secretion by a Janus kinase-signal transducer and activator of transcription (JAK-STAT)-dependent mechanism. In the current study, we examined the regulation of NNT-1/BSF-3 mRNA expression in murine pituitary folliculostellate TtT/GF cells using Northern blot technique. A 5- to 9-fold and a 4- to 7-fold induction in NNT-1/BSF-3 mRNA expression was observed between 2 and 6 h stimulation with the protein kinase C (PKC) stimulus phorbol-12-myristate-13-acetate (100 nm) and the protein kinase A (PKA) stimulus Bu(2)cAMP (5 mm), respectively. Pituitary adenylate cyclase-activating polypeptide (PACAP-38, 50 nm) and vasoactive intestinal peptide (VIP, 50 nm) also stimulated NNT-1/BSF-3 mRNA expression 5- to 9-fold between 2 and 6 h. Preincubation with PKC and PKA inhibitors such as H-7 (20 microm), GF109203X (50 microm), and H-89 (50 microm) decreased the stimulatory effects of PACAP and VIP. Both PACAP-38 and VIP also rapidly induced ERK1/2 phosphorylation and their stimulatory effect on NNT-1/BSF-3 mRNA expression was reduced by the MAPK kinase/ERK kinase (MEK) inhibitor U0126 (10 microm). Dexamethasone (10(-7) m) was a potent inhibitor of phorbol-12-myristate-13-acetate-induced NNT-1/BSF-3 expression. RT-PCR analysis demonstrated TtT/GF cells to express the short and the hop variant but not the hip variant of the PACAP-1 receptor (PAC1-R). In addition, TtT/GF cells express the VIP/PACAP-2 receptor (VPAC2-R). In summary, NNT-1/BSF-3 is expressed in pituitary folliculostellate TtT/GF cells and induced by PKC-, PKA-, and ERK1/2-dependent mechanisms. The novel gp130 cytokine NNT-1/BSF-3 derived from folliculostellate cells might act as a paracrine neuroimmunoendocrine modulator of pituitary corticotroph function.

  14. 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

  15. Nucleotidyl cyclase activity of soluble guanylyl cyclase in intact cells.

    PubMed

    Bähre, Heike; Danker, Kerstin Y; Stasch, Johannes-Peter; Kaever, Volkhard; Seifert, Roland

    2014-01-24

    Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and generates the second messenger cyclic GMP (cGMP). Recently, purified sGC α1β1 has been shown to additionally generate the cyclic pyrimidine nucleotides cCMP and cUMP. However, since cyclic pyrimidine nucleotide formation occurred only the presence of Mn(2+) but not Mg(2+), the physiological relevance of these in vitro findings remained unclear. Therefore, we studied cyclic nucleotide formation in intact cells. We observed NO-dependent cCMP- and cUMP formation in intact HEK293 cells overexpressing sGC α1β1 and in RFL-6 rat fibroblasts endogenously expressing sGC, using HPLC-tandem mass spectrometry. The identity of cCMP and cUMP was unambiguously confirmed by HPLC-time-of-flight mass spectrometry. Our data indicate that cCMP and cUMP play second messenger roles and that Mn(2+) is a physiological sGC cofactor. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Coupled ATPase-adenylate kinase activity in ABC transporters.

    PubMed

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

    2016-12-22

    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 (31)P 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.

  17. 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

  18. 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.

  19. 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

  20. Pituitary adenylate cyclase activating polypeptide (PACAP), stress, and sex hormones.

    PubMed

    King, S Bradley; Toufexis, Donna J; Hammack, Sayamwong E

    2017-06-14

    Stressor exposure is associated with the onset and severity of many psychopathologies that are more common in women than men. Moreover, the maladaptive expression and function of stress-related hormones have been implicated in these disorders. Evidence suggests that PACAP has a critical role in the stress circuits mediating stress-responding, and PACAP may interact with sex hormones to contribute to sex differences in stress-related disease. In this review, we describe the role of the PACAP/PAC1 system in stress biology, focusing on the role of stress-induced alterations in PACAP expression and signaling in the development of stress-induced behavioral change. Additionally, we present more recent data suggesting potential interactions between stress, PACAP, and circulating estradiol in pathological states, including PTSD. These studies suggest that the level of stress and circulating gonadal hormones may differentially regulate the PACAPergic system in males and females to influence anxiety-like behavior and may be one mechanism underlying the discrepancies in human psychiatric disorders.

  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. 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.

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

    PubMed

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

    2017-06-01

    The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, αMβ2 integrin, or CD11b/CD18). CyaA delivers into cells an N-terminal adenylyl cyclase (AC) enzyme domain that is activated by cytosolic calmodulin and catalyzes unregulated conversion of cellular ATP into cyclic AMP (cAMP), a key second messenger subverting bactericidal activities of phagocytes. In parallel, the hemolysin (Hly) moiety of CyaA forms cation-selective hemolytic pores that permeabilize target cell membranes. We constructed the first B. pertussis mutant secreting a CyaA toxin having an intact capacity to deliver the AC enzyme into CD11b-expressing (CD11b(+)) host phagocytes but impaired in formation of cell-permeabilizing pores and defective in cAMP elevation in CD11b(-) cells. The nonhemolytic AC(+) Hly(-) bacteria inhibited the antigen-presenting capacities of coincubated mouse dendritic cells in vitro and skewed their Toll-like receptor (TLR)-triggered maturation toward a tolerogenic phenotype. The AC(+) Hly(-) mutant also infected mouse lungs as efficiently as the parental AC(+) Hly(+) strain. Hence, elevation of cAMP in CD11b(-) cells and/or the pore-forming capacity of CyaA were not required for infection of mouse airways. The latter activities were, however, involved in bacterial penetration across the epithelial layer, enhanced neutrophil influx into lung parenchyma during sublethal infections, and the exacerbated lung pathology and lethality of B. pertussis infections at higher inoculation doses (>10(7) CFU/mouse). The pore-forming activity of CyaA further synergized with the cAMP-elevating activity in downregulation of major histocompatibility complex class II (MHC-II) molecules on infiltrating myeloid cells, likely contributing to immune subversion of host defenses by the whooping cough agent. Copyright © 2017 American Society for Microbiology.

  4. Targeted mutations that ablate either the adenylate cyclase or hemolysin function of the bifunctional cyaA toxin of Bordetella pertussis abolish virulence.

    PubMed Central

    Gross, M K; Au, D C; Smith, A L; Storm, D R

    1992-01-01

    Bordetella pertussis, the causative agent of whooping cough, secretes several toxins implicated in this disease. One of these putative virulence factors is the adenylate cyclase (AC) toxin that elevates intracellular cAMP in eukaryotic cells to cytotoxic levels. This toxin is a bifunctional protein comprising both AC and hemolysin (HLY) enzymatic domains. The gene encoding the AC toxin (cyaA) is expressed as part of an operon that includes genes required for secretion or activation of the toxin. Because of this genetic organization, it is difficult to create B. pertussis mutants of cyaA that are ablations of a single enzyme function by conventional means, such as transposon mutagenesis. Therefore, to clarify the role of individual toxin functions in the virulence of B. pertussis, we have used site-directed or deletion mutagenesis and genetic recombination to specifically target the cyaA gene of B. pertussis to produce mutants that lack only the AC or HLY activity of this toxin. A point mutant of B. pertussis with abolished AC catalytic activity was greater than 1000 times less pathogenic to newborn mice than wild-type bacteria, directly demonstrating the importance of the AC toxin in pertussis virulence. Similarly, an in-frame deletion mutant of B. pertussis that lacks HLY is equally avirulent, supporting observations that the HLY domain plays a critical role in AC toxin entry into cells. Furthermore, the genetically inactivated AC toxin produced by the point mutant is antigenically similar to the native toxin, suggesting that this strain may be useful in the development of pertussis component vaccines. Images PMID:1594590

  5. 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

  6. Catecholamine-induced desensitization of turkey erythrocyte adenylate cyclase. Structural alterations in the beta-adrenergic receptor revealed by photoaffinity labeling.

    PubMed

    Stadel, J M; Nambi, P; Lavin, T N; Heald, S L; Caron, M G; Lefkowitz, R J

    1982-08-25

    Preincubation of turkey erythrocytes with isoproterenol results in an impaired ability of beta-adrenergic agonists to stimulate adenylate cyclase in membranes prepared from these cells. The biochemical basis for this agonist-induced desensitization was investigated using the new beta-adrenergic antagonist photoaffinity label [125I]p-azidobenzylcarazolol ([125I]PABC). Exposure of [125I]PABC-labeled turkey erythrocyte membranes to high intensity light leads to specific covalent incorporation of the labeled compound into two polypeptides, Mr approximately equal to 38,000 and 50,000, as determined by sodium dodecyl sulfate-polyacrylamide electrophoresis. Incorporation of [125I]PABC into these two polypeptides is completely blocked by a beta-adrenergic agonist and antagonist consistent with covalent labeling of the beta-adrenergic receptor. After desensitization of the turkey erythrocyte by preincubation with 10(-5) M isoproterenol, the beta-adrenergic receptor polypeptides specifically labeled by [125I]PABC in membranes prepared from desensitized erythrocytes were of larger apparent molecular weight (Mr approximately equal to 42,000 versus 38,000, and 53,000 versus 50,000) compared to controls. When included during the preincubation of the erythrocytes with isoproterenol, the antagonist propranolol (10(-5) M) inhibited both agonist-promoted desensitization of the adenylate cyclase and the altered mobility of the [125I]PABC-labeled receptor polypeptides. These data indicate that structural alterations in the beta-adrenergic receptor accompany the desensitization process in turkey erythrocytes.

  7. 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.

  8. Role of CD11b/CD18 in the Process of Intoxication by the Adenylate Cyclase Toxin of Bordetella pertussis

    PubMed Central

    Eby, Joshua C.; Gray, Mary C.; Mangan, Annabelle R.; Donato, Gina M.

    2012-01-01

    The adenylate cyclase toxin (ACT) of Bordetella pertussis does not require a receptor to generate intracellular cyclic AMP (cAMP) in a broad range of cell types. To intoxicate cells, ACT binds to the cell surface, translocates its catalytic domain across the cell membrane, and converts intracellular ATP to cAMP. In cells that express the integrin CD11b/CD18 (CR3), ACT is more potent than in CR3-negative cells. We find, however, that the maximum levels of cAMP accumulation inside CR3-positive and -negative cells are comparable. To better understand how CR3 affects the generation of cAMP, we used Chinese hamster ovary and K562 cells transfected to express CR3 and examined the steps in intoxication in the presence and absence of the integrin. The binding of ACT to cells is greater in CR3-expressing cells at all concentrations of ACT, and translocation of the catalytic domain is enhanced by CR3 expression, with ∼80% of ACT molecules translocating their catalytic domain in CR3-positive cells but only 25% in CR3-negative cells. Once in the cytosol, the unregulated catalytic domain converts ATP to cAMP, and at ACT concentrations >1,000 ng/ml, the intracellular ATP concentration is <5% of that in untreated cells, regardless of CR3 expression. This depletion of ATP prevents further production of cAMP, despite the CR3-mediated enhancement of binding and translocation. In addition to characterizing the effects of CR3 on the actions of ACT, these data show that ATP consumption is yet another concentration-dependent activity of ACT that must be considered when studying how ACT affects target cells. PMID:22144488

  9. 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.

  10. 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

  11. 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.

  12. X-linked recessive congenital muscle fiber hypotrophy with central nuclei: abnormalities of growth and adenylate cyclase in muscle tissue cultures.

    PubMed

    Askanas, V; Engel, W K; Reddy, N B; Barth, P G; Bethlem, J; Krauss, D R; Hibberd, M E; Lawrence, J V; Carter, L S

    1979-10-01

    Muscle cells in cultures established from biopsy specimens of two children with an infantile-fatal form of X-linked recessive muscle fiber smallness with central nuclei showed an unusual ability to proliferate through numerous passages. Ultrastructurally, the cultured muscle fibers appeared very immature even after several weeks. The nuclei were large, the number of ribosomes was greatly increased, the myofibrils remained unstriated, and glycogen was accumulated in large lakes. The plasmalemma bound concanavalin A, alpha-bungarotoxin, and ruthenium red normally, but with tannic acid it did not show the dark binding of mature fibers. Biochemically, in the cultured muscle fibers, beta-adrenergic receptors were quantitatively normal. The level of adenylate cyclase in membranes was less than in cultured normal muscle; this defect could be responsible for impaired control mechanisms resulting in the other abnormalities observed.

  13. 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.

  14. Adenylate cyclase 1 (ADCY1) mutations cause recessive hearing impairment in humans and defects in hair cell function and hearing in zebrafish

    PubMed Central

    Santos-Cortez, Regie Lyn P.; Lee, Kwanghyuk; Giese, Arnaud P.; Ansar, Muhammad; Amin-Ud-Din, Muhammad; Rehn, Kira; Wang, Xin; Aziz, Abdul; Chiu, Ilene; Hussain Ali, Raja; Smith, Joshua D.; Shendure, Jay; Bamshad, Michael; Nickerson, Deborah A.; Ahmed, Zubair M.; Ahmad, Wasim; Riazuddin, Saima; Leal, Suzanne M.

    2014-01-01

    Cyclic AMP (cAMP) production, which is important for mechanotransduction within the inner ear, is catalyzed by adenylate cyclases (AC). However, knowledge of the role of ACs in hearing is limited. Previously, a novel autosomal recessive non-syndromic hearing impairment locus DFNB44 was mapped to chromosome 7p14.1-q11.22 in a consanguineous family from Pakistan. Through whole-exome sequencing of DNA samples from hearing-impaired family members, a nonsense mutation c.3112C>T (p.Arg1038*) within adenylate cyclase 1 (ADCY1) was identified. This stop-gained mutation segregated with hearing impairment within the family and was not identified in ethnically matched controls or within variant databases. This mutation is predicted to cause the loss of 82 amino acids from the carboxyl tail, including highly conserved residues within the catalytic domain, plus a calmodulin-stimulation defect, both of which are expected to decrease enzymatic efficiency. Individuals who are homozygous for this mutation had symmetric, mild-to-moderate mixed hearing impairment. Zebrafish adcy1b morphants had no FM1-43 dye uptake and lacked startle response, indicating hair cell dysfunction and gross hearing impairment. In the mouse, Adcy1 expression was observed throughout inner ear development and maturation. ADCY1 was localized to the cytoplasm of supporting cells and hair cells of the cochlea and vestibule and also to cochlear hair cell nuclei and stereocilia. Ex vivo studies in COS-7 cells suggest that the carboxyl tail of ADCY1 is essential for localization to actin-based microvilli. These results demonstrate that ADCY1 has an evolutionarily conserved role in hearing and that cAMP signaling is important to hair cell function within the inner ear. PMID:24482543

  15. 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

  16. 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.

  17. 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.

  18. Guanylyl cyclase-D in the olfactory CO2 neurons is activated by bicarbonate.

    PubMed

    Sun, Liming; Wang, Huayi; Hu, Ji; Han, Jinlong; Matsunami, Hiroaki; Luo, Minmin

    2009-02-10

    Atmospheric CO(2) is an important environmental cue that regulates several types of animal behavior. In mice, CO(2) responses of the olfactory sensory neurons (OSNs) require the activity of carbonic anhydrase to catalyze the conversion of CO(2) to bicarbonate and the opening of cGMP-sensitive ion channels. However, it remains unknown how the enhancement of bicarbonate levels results in cGMP production. Here, we show that bicarbonate activates cGMP-producing ability of guanylyl cyclase-D (GC-D), a membrane GC exclusively expressed in the CO(2)-responsive OSNs, by directly acting on the intracellular cyclase domain of GC-D. Also, the molecular mechanism for GC-D activation is distinct from the commonly believed model of "release from repression" for other membrane GCs. Our results contribute to our understanding of the molecular mechanisms of CO(2) sensing and suggest diverse mechanisms of molecular activation among membrane GCs.

  19. Control of guanylate cyclase activity in the rod outer segment.

    PubMed

    Pannbacker, R G

    1973-12-14

    Mammalian photoreceptors contain a guanylate cyclase which has a high specific activity and is inhibited by exposure of the rod outer segment to light. Several minutes are required for this inhibition to take effect, indicating that it is not a step in visual excitation. The activity of the enzyme is sensitive to the concentration of calcium ion in the medium, suggesting that light-induced changes in calcium distribution in the photoreceptor could control guanylate cyclase activity.

  20. Inhibitory role of monovalent ions on rat brain cortex adenylyl cyclase activity.

    PubMed

    Nikolic, Ivana; Mitrovic, Marina; Zelen, Ivanka; Zaric, Milan; Kastratovic, Tatjana; Stanojevic, Marijana; Nenadovic, Milutin; Stojanovic, Tomislav

    2013-10-01

    Adenylyl cyclases, comprise of a large family of enzymes that catalyze synthesis of the cyclic AMP from ATP. The aim of our study was to determine the effect of monovalent ions on both basal, stimulated adenylate cyclase EC 4.6.1.1 (AC) activity and C unit of AC and on GTPase active G-protein in the synaptic membranes of rat brain cortex. The effect of ion concentration from 30 to 200 mM (1 mM MgCl2) showed dose-dependent and significant inhibition of the basal AC activity, stimulated and unstimulated C unit activity. Stimulation of AC with 5 μM GTPγS in the presence of 50-200 mM of tested salts showed inhibitory effect on the AC activity. From our results it could be postulated that the investigated monovalent ions exert inhibitory effect on the AC complex activity by affecting the intermolecular interaction of the activated α subunit of G/F protein and the C unit of AC complex an inhibitory influence of tested monovalent ions on these molecular interaction.

  1. Measurement of Nonribosomal Peptide Synthetase Adenylation Domain Activity Using a Continuous Hydroxylamine Release Assay.

    PubMed

    Duckworth, Benjamin P; Wilson, Daniel J; Aldrich, Courtney C

    2016-01-01

    Adenylation is a crucial enzymatic process in the biosynthesis of nonribosomal peptide synthetase (NRPS) derived natural products. Adenylation domains are considered the gatekeepers of NRPSs since they select, activate, and load the carboxylic acid substrate onto a downstream peptidyl carrier protein (PCP) domain of the NRPS. We describe a coupled continuous kinetic assay for NRPS adenylation domains that substitutes the PCP domain with hydroxylamine as the acceptor molecule. The pyrophosphate released from the first-half reaction is then measured using a two-enzyme coupling system, which detects conversion of the chromogenic substrate 7-methylthioguanosine (MesG) to 7-methylthioguanine. From profiling substrate specificity of unknown or engineered adenylation domains to studying chemical inhibition of adenylating enzymes, this robust assay will be of widespread utility in the broad field NRPS enzymology.

  2. Alzheimer amyloid beta-peptide A-beta25-35 blocks adenylate cyclase-mediated forms of hippocampal long-term potentiation.

    PubMed

    Bisel, Blaine E; Henkins, Kristen M; Parfitt, Karen D

    2007-02-01

    Progressive memory loss and deposition of amyloid beta (Abeta) peptides throughout cortical regions are hallmarks of Alzheimer's disease (AD). Several studies in mice and rats have shown that overexpression of amyloid precursor protein (APP) or pretreatment with Abeta peptide fragments results in the inhibition of hippocampal long-term potentiation (LTP) as well as impairments in learning and memory of hippocampal-dependent tasks. For these studies we have investigated the effects of the Abeta(25-35) peptide fragment on LTP induced by adenylate cyclase stimulation followed immediately by application of Mg(++)-free aCSF ("chemLTP"). Treatment of young adult slices with the Abeta(25-35) peptide had no significant effect on basal synaptic transmission in area CA1, but treatment with the peptide for 20 min before inducing chemLTP with isoproterenol (ISO; 1 microM) or forskolin (FSK;10 microM) + Mg(++)-free aCSF resulted in complete blockade of LTP. In contrast, normal ISO-chemLTP was observed after treatment with the control peptide Abeta(35-25). The ability of the Abeta(25-35) peptide fragment to block this and other forms of synaptic plasticity may help elucidate the mechanisms underlying hippocampal deficits observed in animal models of AD and/or AD individuals.

  3. Ca2+ Influx and Tyrosine Kinases Trigger Bordetella Adenylate Cyclase Toxin (ACT) Endocytosis. Cell Physiology and Expression of the CD11b/CD18 Integrin Major Determinants of the Entry Route

    PubMed Central

    Etxebarria, Aitor; González-Bullón, David; Gómez-Bilbao, Geraxane; Ostolaza, Helena

    2013-01-01

    Humans infected with Bordetella pertussis, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT) which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3), its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca2+ influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface. PMID:24058533

  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. 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.

  6. 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.

  7. [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.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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

  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. 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.

  17. Nucleotidyl cyclase activity of particulate guanylyl cyclase A: comparison with particulate guanylyl cyclases E and F, soluble guanylyl cyclase and bacterial adenylyl cyclases CyaA and edema factor.

    PubMed

    Beste, Kerstin Y; Spangler, Corinna M; Burhenne, Heike; Koch, Karl-Wilhelm; Shen, Yuequan; Tang, Wei-Jen; Kaever, Volkhard; Seifert, Roland

    2013-01-01

    Guanylyl cyclases (GCs) regulate many physiological processes by catalyzing the synthesis of the second messenger cGMP. The GC family consists of seven particulate GCs (pGCs) and a nitric oxide-activated soluble GC (sGC). Rat sGC α1β1 possesses much broader substrate specificity than previously assumed. Moreover, the exotoxins CyaA from Bordetella pertussis and edema factor (EF) from Bacillus anthracis possess nucleotidyl cyclase (NC) activity. pGC-A is a natriuretic peptide-activated homodimer with two catalytic sites that act cooperatively. Here, we studied the NC activity of rat pGC-A in membranes of stably transfected HEK293 cells using a highly sensitive and specific HPLC-MS/MS technique. GTP and ITP were effective, and ATP and XTP were only poor, pGC-A substrates. In contrast to sGC, pGC-A did not use CTP and UTP as substrates. pGC-E and pGC-F expressed in bovine rod outer segment membranes used only GTP as substrate. In intact HEK293 cells, pGC-A generated only cGMP. In contrast to pGCs, EF and CyaA showed very broad substrate-specificity. In conclusion, NCs exhibit different substrate-specificities, arguing against substrate-leakiness of enzymes and pointing to distinct physiological functions of cyclic purine and pyrimidine nucleotides.

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-01

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

  20. 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

  1. Alteration of the Cytotoxic Action of Sensitized Lymphocytes by Cholinergic Agents and Activators of Adenylate Cyclase

    PubMed Central

    Strom, Terry B.; Deisseroth, Albert; Morganroth, Joel; Carpenter, Charles B.; Merrill, John P.

    1972-01-01

    The cytotoxic action of lymphocytes upon cells bearing alloantigens to which they are sensitized is inhibited by agents that elevate intracellular amounts of 3′:5′-cyclic AMP: prostaglandin E1, cholera toxin, and theophylline. Cholinergic agents, added in the range of 1 to 100 pM, enhance cytotoxicity, an effect that is blocked by atropine. Because cholinergic agents elevate cyclic GMP in other in vitro systems, these findings suggest that the cytotoxic process effected by sensitized lymphocytes is a secretory phenomenon modulated by cyclic AMP and cyclic GMP. PMID:4342971

  2. 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

  3. Activity of guanylyl cyclase activators on the reaction of tracheal smooth muscle contraction.

    PubMed

    Glaza, Izabela; Szadujkis-Szadurski, Leszek; Szadujkis-Szadurski, Rafał; Gajdus, Marta; Rzepka, Alicja; Gurtowska, Natalia

    2011-08-05

    The subject of the study compare the influences of YC-1 guanylyl cyclase activator with ODQ guanylyl cyclase inhibitor on the tracheal smooth muscle contraction induced by carbachol. The study specified the influence of increasing concentrations of soluble guanylyl cyclase activators YC-1 and 8Br cGMP on the reaction of tracheal smooth muscle contraction released by carbachol. The author also examined the effect of increasing concentrations of soluble guanylyl cyclase inhibitor ODQ on the concentration-effect curves for carbachol. Testing was conducted on an isolated trachea of both sexes of Wistar rats with weight ranging between 350 g and 450 g. Tracheas were prepared in accordance with the Akcasu (1959) method in Szadujkis-Szadurski (1996) modification. Concentration-effect curves were determined with the use of cumulated concentration method, in accordance with the van Rossum method (1963) in Kenakin (2006) modification. According to conducted testing, activation of soluble guanylyl cyclase with the use of YC-1 and 8Br cGMP caused reduced reaction of the tracheal smooth muscle with carbachol on average to 80%. Comparing concentration-effect curves for carbachol before and after the use of 8Br cGMP, similar results were obtained for those released by YC-1. On the other hand, increasing concentrations of guanylyl cyclase inhibitor - ODQ cause shift of curves to the left, decrease of EC(50) value and an increase of maximum reaction to carbachol. Carbachol, depending on concentration, causes tracheal smooth muscle contraction. According to testing, we can confirm that activation of guanylyl cyclase leads to reduction of the reaction of tracheal smooth muscle to carbachol on average up to 80%

  4. beta-Adrenoceptor density and adenylyl cyclase activity in obese rabbit hearts.

    PubMed

    Carroll, J F; Kyser, C K; Martin, M M

    2002-05-01

    To determine whether decreased cardiac responsiveness to isoproterenol in obesity is associated with alterations in beta-receptors and/or adenylyl cyclase activity. ANIMALS AND DESIGN: After 12 weeks of control or ad libitum high-fat diets, left ventricular tissue from lean and obese female New Zealand white rabbits was assayed for beta-receptor binding density (11 lean, 11 obese) and isoproterenol-stimulated adenylyl cyclase activity (eight lean, 10 obese). Nonlinear least squares regression analysis was used to determine maximum density of beta-receptors and receptor affinity for (125)I-iodocyanopindolol. Four-parameter logistic regression was used to determine minimum, maximum, slope and EC(50) for isoproterenol-stimulated adenylyl cyclase activity. Obese rabbits had elevated resting blood pressure and heart rate, and higher ventricular weights. However, beta-adrenoceptor density and affinity were not significantly different in lean and obese rabbits. Basal and maximum isoproterenol-stimulated adenylyl cyclase activity did not differ between lean and obese rabbits. In addition, maximal stimulation in response to sodium flouride did not differ between lean and obese. EC(50) for isoproterenol-stimulated adenylyl cyclase activity did not differ between lean and obese rabbits. Obesity-related decreases in responsiveness of the isolated heart to isoproterenol are not associated with alterations in beta-receptor density and affinity. In addition, adenylyl cyclase activity appeared unchanged in ventricular preparations from obese rabbits. Decreased responsiveness to isoproterenol in obesity may be due to defects downstream of adenylyl cyclase activation of cyclic AMP.

  5. Soluble guanylyl cyclase activators increase the expression of tolerance to morphine analgesic effect.

    PubMed

    Durmus, N; Bagcivan, I; Ozdemir, E; Altun, A; Gursoy, S

    2014-01-01

    It is aimed to investigate the effects of guanylyl cyclase activation and inhibition on acute morphine antinociception and the development of tolerance to its effect. Nitric oxide-soluble guanylyl cyclase signal transduction cascade suggested to play an important role in the development of tolerance to antinociceptive effects of morphine. Nociception was evaluated by tail flick and hot plate tests in male Wistar rats. The analgesic effects of intraperitoneal protoporphyrin IX (PPIX; an activator of soluble guanylyl cyclase), 3-morpholinosydnonimine hydrochloride (SIN-1; NO donor and activator of guanylyl cyclase), S-Nitroso-N-acetylpenicillamine (SNAP; an activator of guanylyl cyclase), 3,3-Bis (amino ethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18; NO donor activating guanylyl cyclase) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; an inhibitor of guanylyl cyclase) alone or in combination with subcutaneous morphine injection were evaluated. Their effects on morphine tolerance development were evaluated by giving these agents 20 minutes prior to twice daily morphine injection during tolerance development for 5 days. On day 6, the expression of morphine tolerance was determined. PPIX, SIN-1, SNAP and NOC-18 significantly increased expression of morphine tolerance while ODQ decreased. These data suggested that sGC activators have a significant role in tolerance to the analgesic effect of morphine (Tab. 1, Fig. 4, Ref. 29).

  6. Activity of squalene-hopene cyclases in bicontinuous microemulsions.

    PubMed

    Steudle, Anne K; Nestl, Bettina M; Hauer, Bernhard; Stubenrauch, Cosima

    2015-11-01

    The paper at hand deals with biocatalysis in bicontinuous microemulsions. The latter consist of a dynamic network of oil and water domains separated by a monolayer of surfactant molecules, i.e. the interfacial layer. A microemulsion with the composition buffer--n-octane--nonionic surfactant was tested as reaction medium for an enzyme-catalysed reaction with a focus on the conversion of hydrophobic substrates, which are difficult to convert in aqueous buffer solutions. For the study at hand, we chose to investigate the activity of the squalene-hopene cyclase from Alicyclobacillus acidocaldarius (AacSHC) towards its natural substrate squalene in bicontinuous microemulsions. Firstly, the study revealed that the activity of AacSHC depends linearly on the enzyme concentration. Secondly, a hyperbolic curve was found for the dependence of the activity on the substrate concentration and a saturation of the AacSHC at substrate concentrations above 20mM was observed. Thirdly, the composition of the interfacial layer was found to have no significant influence on the activity or on the conformation of AacSHC. Surprisingly and unexpectedly, a distinctly enhanced selectivity towards hopene was discovered in the microemulsion. To conclude, bicontinuous microemulsions were found to be a suitable reaction medium for biocatalytic reactions with the enzyme AacSHC. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Bicarbonate activation of adenylyl cyclase via promotion of catalytic active site closure and metal recruitment.

    PubMed

    Steegborn, Clemens; Litvin, Tatiana N; Levin, Lonny R; Buck, Jochen; Wu, Hao

    2005-01-01

    In an evolutionarily conserved signaling pathway, 'soluble' adenylyl cyclases (sACs) synthesize the ubiquitous second messenger cyclic adenosine 3',5'-monophosphate (cAMP) in response to bicarbonate and calcium signals. Here, we present crystal structures of a cyanobacterial sAC enzyme in complex with ATP analogs, calcium and bicarbonate, which represent distinct catalytic states of the enzyme. The structures reveal that calcium occupies the first ion-binding site and directly mediates nucleotide binding. The single ion-occupied, nucleotide-bound state defines a novel, open adenylyl cyclase state. In contrast, bicarbonate increases the catalytic rate by inducing marked active site closure and recruiting a second, catalytic ion. The phosphates of the bound substrate analogs are rearranged, which would facilitate product formation and release. The mechanisms of calcium and bicarbonate sensing define a reaction pathway involving active site closure and metal recruitment that may be universal for class III cyclases.

  8. 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.

  9. Modulation of soluble guanylate cyclase activity by phosphorylation.

    PubMed

    Murthy, Karnam S

    2004-11-01

    The levels of the cGMP in smooth muscle of the gut reflect continued synthesis by soluble guanylate cyclase (GC) and breakdown by phosphodiesterase 5 (PDE5). Soluble GC is a haem-containing, heterodimeric protein consisting alpha- and beta-subunits: each subunit has N-terminal regulatory domain and a C-terminal catalytic domain. The haem moiety acts as an intracellular receptor for nitric oxide (NO) and determines the ability of NO to activate the enzyme and generate cGMP. In the present study the mechanism by which protein kinases regulate soluble GC in gastric smooth muscle was examined. Sodium nitroprusside (SNP) acting as a NO donor stimulated soluble GC activity and increased cGMP levels. SNP induced soluble GC phosphorylation in a concentration-dependent fashion. SNP-induced soluble GC phosphorylation was abolished by the selective cGMP-dependent protein kinase (PKG) inhibitors, Rp-cGMPS and KT-5823. In contrast, SNP-stimulated soluble GC activity and cGMP levels were significantly enhanced by Rp-cGMPS and KT-5823. Phosphorylation and inhibition of soluble GC were PKG specific, as selective activator of cAMP-dependent protein kinase, Sp-5, 6-DCl-cBiMPS had no effect on SNP-induced soluble GC phosphorylation and activity. The ability of PKG to stimulate soluble GC phosphorylation was demonstrated in vitro by back phosphorylation technique. Addition of purified phosphatase 1 inhibited soluble GC phosphorylation in vitro, and inhibition was reversed by a high concentration (10 microM) of okadaic acid. In gastric smooth muscle cells, inhibition of phosphatase activity by okadaic acid increased soluble GC phosphorylation in a concentration-dependent fashion. The increase in soluble GC phosphorylation inhibited SNP-stimulated soluble GC activity and cGMP formation. The results implied the feedback inhibition of soluble GC activity by PKG-dependent phosphorylation impeded further formation of cGMP.

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

    PubMed

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

    2008-04-01

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

  11. 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

  12. 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.

  13. The PI3K-mediated activation of CRAC independently regulates adenylyl cyclase activation and chemotaxis.

    PubMed

    Comer, Frank I; Lippincott, Christopher K; Masbad, Joseph J; Parent, Carole A

    2005-01-26

    The ability of a cell to detect an external chemical signal and initiate a program of directed migration along a gradient comprises the fundamental process called chemotaxis. Investigations in Dictyostelium discoideum and neutrophils have established that pleckstrin homology (PH) domain-containing proteins that bind to the PI3K products PI(3,4)P2 and PI(3,4,5)P3, such as CRAC (cytosolic regulator of adenylyl cyclase) and Akt/PKB, translocate specifically to the leading edge of chemotaxing cells. CRAC is essential for the chemoattractant-mediated activation of the adenylyl cyclase ACA, which converts ATP into cAMP, the primary chemoattractant for D. discoideum. The mechanisms by which CRAC activates ACA remain to be determined. We now show that in addition to its essential role in the activation of ACA, CRAC is involved in regulating chemotaxis. Through mutagenesis, we show that these two functions are independently regulated downstream of PI3K. A CRAC mutant that has lost the capacity to bind PI3K products does not support chemotaxis and shows minimal ACA activation. Finally, overexpression of CRAC and various CRAC mutants show strong effects on ACA activation with little effect on chemotaxis. These findings establish that chemoattractant-mediated activation of PI3K is important for the CRAC-dependent regulation of both chemotaxis and adenylyl cyclase activation.

  14. Formycin triphosphate as a probe for the ATP binding site involved in the activation of guanylate cyclase.

    PubMed

    Chang, C H; Yu, Z N; Song, D L

    1992-10-01

    Formycin A triphosphate (FTP), a fluorescent analog of ATP, slightly increased basal guanylate cyclase activity, but significantly potentiated guanylate cyclase activity stimulated by atrial natriuretic factor (ANF) in rat lung membranes. FTP potentiated ANF-stimulated guanylate cyclase activity with an EC50 at about 90 microM and inhibited ATP-stimulated guanylate cyclase activity with an IC50 at about 100 microM. These results indicate that FTP binds more tightly than ATP for the same binding site. Therefore, FTP would be an excellent tool for studying the ATP binding site.

  15. Active-site modifications of adenylation domains lead to hydrolysis of upstream nonribosomal peptidyl thioester intermediates.

    PubMed

    Uguru, Gabriel C; Milne, Claire; Borg, Matthew; Flett, Fiona; Smith, Colin P; Micklefield, Jason

    2004-04-28

    Site-directed mutagenesis of nonribosomal peptide synthetase (NRPS) adenylation (A) domains was investigated as a means to engineer new calcium-dependent antibiotics (CDA) in Streptomyces coelicolor. Single- and double-point mutants of the CDA NRPS module 7, A-domain were generated, which were predicted to alter the specificity of this domain from Asp to Asn. The double-point mutant produced a new peptide CDA2a-7N containing Asn at position 7 as expected. However, in both the single- and the double-point mutants, significant hydrolysis of the CDA-6mer intermediate was evident. One explanation for this is that the mutant module 7 A-domain activates Asn instead of Asp; however, the Asn-thioester intermediate is only weakly recognized by the upstream C-domain acceptor site (a), allowing a water molecule to intercept the hexapeptidyl intermediate in the donor site (d).

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

    PubMed Central

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

    2003-01-01

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

  17. [The role of sulfhydryl groups in functioning of components of adenylate cyclase signal system in smooth muscles of the mollusk Anodonta cygnea (effect of N-ethylmaleimide and p-chloromercuribenzoic acid)].

    PubMed

    Shpakov, A O; Derkach, K V

    1999-01-01

    The alkylating agent N-ethylameimide and the sulfhydryl group blocker p-chloromercuribenzoic acid (CPMA) inhibited in dose-dependent manner both basal activity of adenylyl cyclase (AC) and its activity stimulated by non-hormonal substances (forskolin, sodium fluoride, guanylilimidodiphosphate) in smooth muscles of the freshwater bivalve mollusk Anodonta cygnea. The double increase (from 30 to 60 min) in the time of preincubation of a sarcolemmal membrane fraction with ethylmaleimide and CPMA led to an essential increase in enzyme inhibition (especially for CPMA). 50 mM SH-containing reagent beta-mercaptoethanol (ME) partially restored the AC activity, inhibited by N-ethylmaleimide and CPMA, except when these two latter reagents were in high concentrations (1-10 and 0.5 mM, respectively). The data obtained point to the key role of cysteine SH-groups in regulation of the functional activity of proteins, components of the adenylyl cyclase system--AC and heterotrimeric G-proteins.

  18. Evaluating the role of retinal membrane guanylyl cyclase 1 (RetGC1) domains in binding guanylyl cyclase-activating proteins (GCAPs).

    PubMed

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

    2015-03-13

    Retinal membrane guanylyl cyclase 1 (RetGC1) regulated by guanylyl cyclase-activating proteins (GCAPs) controls photoreceptor recovery and when mutated causes blinding disorders. We evaluated the principal models of how GCAP1 and GCAP2 bind RetGC1: through a shared docking interface versus independent binding sites formed by distant portions of the cyclase intracellular domain. At near-saturating concentrations, GCAP1 and GCAP2 activated RetGC1 from HEK293 cells and RetGC2(-/-)GCAPs1,2(-/-) mouse retinas in a non-additive fashion. The M26R GCAP1, which binds but does not activate RetGC1, suppressed activation of recombinant and native RetGC1 by competing with both GCAP1 and GCAP2. Untagged GCAP1 displaced both GCAP1-GFP and GCAP2-GFP from the complex with RetGC1 in HEK293 cells. The intracellular segment of a natriuretic peptide receptor A guanylyl cyclase failed to bind GCAPs, but replacing its kinase homology and dimerization domains with those from RetGC1 restored GCAP1 and GCAP2 binding by the hybrid cyclase and its GCAP-dependent regulation. Deletion of the Tyr(1016)-Ser(1103) fragment in RetGC1 did not block GCAP2 binding to the cyclase. In contrast, substitutions in the kinase homology domain, W708R and I734T, linked to Leber congenital amaurosis prevented binding of both GCAP1-GFP and GCAP2-GFP. Our results demonstrate that GCAPs cannot regulate RetGC1 using independent primary binding sites. Instead, GCAP1 and GCAP2 bind with the cyclase molecule in a mutually exclusive manner using a common or overlapping binding site(s) in the Arg(488)-Arg(851) portion of RetGC1, and mutations in that region causing Leber congenital amaurosis blindness disrupt activation of the cyclase by both GCAP1 and GCAP2. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Evaluating the Role of Retinal Membrane Guanylyl Cyclase 1 (RetGC1) Domains in Binding Guanylyl Cyclase-activating Proteins (GCAPs)*

    PubMed Central

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

    2015-01-01

    Retinal membrane guanylyl cyclase 1 (RetGC1) regulated by guanylyl cyclase-activating proteins (GCAPs) controls photoreceptor recovery and when mutated causes blinding disorders. We evaluated the principal models of how GCAP1 and GCAP2 bind RetGC1: through a shared docking interface versus independent binding sites formed by distant portions of the cyclase intracellular domain. At near-saturating concentrations, GCAP1 and GCAP2 activated RetGC1 from HEK293 cells and RetGC2−/−GCAPs1,2−/− mouse retinas in a non-additive fashion. The M26R GCAP1, which binds but does not activate RetGC1, suppressed activation of recombinant and native RetGC1 by competing with both GCAP1 and GCAP2. Untagged GCAP1 displaced both GCAP1-GFP and GCAP2-GFP from the complex with RetGC1 in HEK293 cells. The intracellular segment of a natriuretic peptide receptor A guanylyl cyclase failed to bind GCAPs, but replacing its kinase homology and dimerization domains with those from RetGC1 restored GCAP1 and GCAP2 binding by the hybrid cyclase and its GCAP-dependent regulation. Deletion of the Tyr1016–Ser1103 fragment in RetGC1 did not block GCAP2 binding to the cyclase. In contrast, substitutions in the kinase homology domain, W708R and I734T, linked to Leber congenital amaurosis prevented binding of both GCAP1-GFP and GCAP2-GFP. Our results demonstrate that GCAPs cannot regulate RetGC1 using independent primary binding sites. Instead, GCAP1 and GCAP2 bind with the cyclase molecule in a mutually exclusive manner using a common or overlapping binding site(s) in the Arg488–Arg851 portion of RetGC1, and mutations in that region causing Leber congenital amaurosis blindness disrupt activation of the cyclase by both GCAP1 and GCAP2. PMID:25616661

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

    PubMed

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

    2015-08-07

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

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

    PubMed Central

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

    2015-01-01

    The photoreceptor-specific proteins guanylyl cyclase-activating proteins (GCAPs) bind and regulate retinal membrane guanylyl cyclase 1 (RetGC1) but not natriuretic peptide receptor A (NPRA). Study of RetGC1 regulation in vitro and its association with fluorescently tagged GCAP in transfected cells showed that R822P substitution in the cyclase dimerization domain causing congenital early onset blindness disrupted RetGC1 ability to bind GCAP but did not eliminate its affinity for another photoreceptor-specific protein, retinal degeneration 3 (RD3). Likewise, the presence of the NPRA dimerization domain in RetGC1/NPRA chimera specifically disabled binding of GCAPs but not of RD3. In subsequent mapping using hybrid dimerization domains in RetGC1/NPRA chimera, multiple RetGC1-specific residues contributed to GCAP binding by the cyclase, but the region around Met823 was the most crucial. Either positively or negatively charged residues in that position completely blocked GCAP1 and GCAP2 but not RD3 binding similarly to the disease-causing mutation in the neighboring Arg822. The specificity of GCAP binding imparted by RetGC1 dimerization domain was not directly related to promoting dimerization of the cyclase. The probability of coiled coil dimer formation computed for RetGC1/NPRA chimeras, even those incapable of binding GCAP, remained high, and functional complementation tests showed that the RetGC1 active site, which requires dimerization of the cyclase, was formed even when Met823 or Arg822 was mutated. These results directly demonstrate that the interface for GCAP binding on RetGC1 requires not only the kinase homology region but also directly involves the dimerization domain and especially its portion containing Arg822 and Met823. PMID:26100624

  2. [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.

  3. Buspirone and gepirone: partial agonists at the 5HT/sub 1/A receptor linked to adenylate cyclase (AC) in rat and guinea pig hippocampal preparations

    SciTech Connect

    Yocca, F.D.; Hyslop, D.K.; Taylor, D.P.; Maayani, S.

    1986-03-01

    The pharmacologic nature of the 5-HT receptor that is negatively linked to AC in membrane preparations from rat and guinea pig (gp) brain in cell culture and in gp hippocampal homogenates positively linked to AC seem to be indistinguishable from the 5HT/sub 1A/ binding site in similar preparations. Affinity values of chemically unrelated but selective drugs for a binding site are useful for taxonomy of functional receptors. The novel anxiolytic drug buspirone (B) and its analog gepirone (G) exhibit selectivity and affinity for spiperone-sensitive (/sup 3/H)-5-HT and (/sup 3/H)-8-OH-DPAT binding sites in gp and rat hippocampus. In the two species tested, B and G were partial agonists (intrinsic activity approx. = 0.5) compared to 5-HT and its potent analog 5-carboxamideotryptamine (5-COAT) at the 5-HT/sub 1A/ receptor linked to AC. The K/sub B/ value of spiperone determined with B and G was indistinguishable from that determined with 5-HT and 5-COAT (20-30 nM). Since B and G exert unique agonist effects at the functional 5HT/sub 1A/ receptor, their structures may be important for identifying chemical groups necessary for recognition and activation of the 5HT/sub 1A/ receptor.

  4. Cu-free cycloaddition for identifying catalytic active adenylation domains of nonribosomal peptide synthetases by phage display.

    PubMed

    Zou, Yekui; Yin, Jun

    2008-10-15

    To engineer the substrate specificities of nonribosomal peptide synthetases (NRPS), we developed a method to display NRPS modules on M13 phages and select catalytically active adenylation (A) domains that would load azide functionalized substrate analogs to the neighboring peptidyl carrier protein (PCP) domains. Biotin conjugated difluorinated cyclooctyne was used for copper free cycloaddition with an azide substituted substrate attached to PCP. Biotin-labeled phages were selected by binding to streptavidin.

  5. 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)

  6. Demonstration of phosphoryl group transfer indicates that the ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) exhibits adenylate kinase activity.

    PubMed

    Randak, Christoph O; Ver Heul, Amanda R; Welsh, Michael J

    2012-10-19

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane-spanning adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter. ABC transporters and other nuclear and cytoplasmic ABC proteins have ATPase activity that is coupled to their biological function. Recent studies with CFTR and two nonmembrane-bound ABC proteins, the DNA repair enzyme Rad50 and a structural maintenance of chromosome (SMC) protein, challenge the model that the function of all ABC proteins depends solely on their associated ATPase activity. Patch clamp studies indicated that in the presence of physiologically relevant concentrations of adenosine 5'-monophosphate (AMP), CFTR Cl(-) channel function is coupled to adenylate kinase activity (ATP+AMP <==> 2 ADP). Work with Rad50 and SMC showed that these enzymes catalyze both ATPase and adenylate kinase reactions. However, despite the supportive electrophysiological results with CFTR, there are no biochemical data demonstrating intrinsic adenylate kinase activity of a membrane-bound ABC transporter. We developed a biochemical assay for adenylate kinase activity, in which the radioactive γ-phosphate of a nucleotide triphosphate could transfer to a photoactivatable AMP analog. UV irradiation could then trap the (32)P on the adenylate kinase. With this assay, we discovered phosphoryl group transfer that labeled CFTR, thereby demonstrating its adenylate kinase activity. Our results also suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for adenylate kinase activity. These biochemical data complement earlier biophysical studies of CFTR and indicate that the ABC transporter CFTR can function as an adenylate kinase.

  7. Differential Calcium Signaling by Cone Specific Guanylate Cyclase-Activating Proteins from the Zebrafish Retina

    PubMed Central

    Scholten, Alexander; Koch, Karl-Wilhelm

    2011-01-01

    Zebrafish express in their retina a higher number of guanylate cyclase-activating proteins (zGCAPs) than mammalians pointing to more complex guanylate cyclase signaling systems. All six zGCAP isoforms show distinct and partial overlapping expression profiles in rods and cones. We determined critical Ca2+-dependent parameters of their functional properties using purified zGCAPs after heterologous expression in E.coli. Isoforms 1–4 were strong, 5 and 7 were weak activators of membrane bound guanylate cyclase. They further displayed different Ca2+-sensitivities of guanylate cyclase activation, which is half maximal either at a free Ca2+ around 30 nM (zGCAP1, 2 and 3) or around 400 nM (zGCAP4, 5 and 7). Zebrafish GCAP isoforms showed also differences in their Ca2+/Mg2+-dependent conformational changes and in the Ca2+-dependent monomer-dimer equilibrium. Direct Ca2+-binding revealed that all zGCAPs bound at least three Ca2+. The corresponding apparent affinity constants reflect binding of Ca2+ with high (≤100 nM), medium (0.1–5 µM) and/or low (≥5 µM) affinity, but were unique for each zGCAP isoform. Our data indicate a Ca2+-sensor system in zebrafish rod and cone cells supporting a Ca2+-relay model of differential zGCAP operation in these cells. PMID:21829700

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

    PubMed Central

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

    2007-01-01

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

  9. Importance of the region around lysine 196 for catalytic activity of adenylyl cyclase from Escherichia coli.

    PubMed

    Amin, N; Peterkofsky, A

    1994-12-09

    Escherichia coli adenylyl cyclase contains no sequence that corresponds to the previously defined ATP/GTP binding consensus (A,G)XXXXGK(S,T). Using a search for lysine residues located adjacent to glycine residues, three regions that were possible candidates for part of the ATP binding site were identified. These were the residues located at positions 59, 90, and 196. A plasmid vector capable of overexpressing the cya gene under the control of the lambda PL promoter was mutated at these three loci to convert those lysine residues to methionine. Assays for catalytic activity of the mutated hyperexpressed proteins revealed that only the mutation at position 196 led to loss of activity. Photoaffinity labeling experiments using 8-azido-ATP provided evidence that the loss of activity was associated with a loss of the capability of the enzyme to bind ATP. A further series of replacement mutations in the hyperexpression vector was created at position 196. Assays of the adenylyl cyclase activity of the mutated proteins showed that replacement of lysine 196 by arginine led to minimal change in the activity. Replacements by histidine, glutamine, or glutamic acid resulted in approximately 10-20-fold reductions in the activity; replacements by methionine, isoleucine, or aspartic acid resulted in total loss of activity. When the mutated forms of the cya gene were expressed under the control of the cya promoter, the activity of the wild-type protein was higher than that of all the mutants, including the arginine replacement mutant. All of the mutants that retained activity also retained the capability of adenylyl cyclase to be stimulated by either inorganic orthophosphate or GTP. A helical wheel analysis of the region of adenylyl cyclase around lysine 196 revealed a structure compatible with an amphipathic helix with one face enriched with basic amino acid residues. Assays for adenylyl cyclase activity of a series of replacement mutations of residues on the hydrophilic face of

  10. Urea-Dependent Adenylate Kinase Activation following Redistribution of Structural States.

    PubMed

    Rogne, Per; Wolf-Watz, Magnus

    2016-10-04

    Proteins are often functionally dependent on conformational changes that allow them to sample structural states that are sparsely populated in the absence of a substrate or binding partner. The distribution of such structural microstates is governed by their relative stability, and the kinetics of their interconversion is governed by the magnitude of associated activation barriers. Here, we have explored the interplay among structure, stability, and function of a selected enzyme, adenylate kinase (Adk), by monitoring changes in its enzymatic activity in response to additions of urea. For this purpose we used a (31)P NMR assay that was found useful for heterogeneous sample compositions such as presence of urea. It was found that Adk is activated at low urea concentrations whereas higher urea concentrations unfolds and thereby deactivates the enzyme. From a quantitative analysis of chemical shifts, it was found that urea redistributes preexisting structural microstates, stabilizing a substrate-bound open state at the expense of a substrate-bound closed state. Adk is rate-limited by slow opening of substrate binding domains and the urea-dependent redistribution of structural states is consistent with a model where the increased activity results from an increased rate-constant for domain opening. In addition, we also detected a strong correlation between the catalytic free energy and free energy of substrate (ATP) binding, which is also consistent with the catalytic model for Adk. From a general perspective, it appears that urea can be used to modulate conformational equilibria of folded proteins toward more expanded states for cases where a sizeable difference in solvent-accessible surface area exists between the states involved. This effect complements the action of osmolytes, such as trimethylamine N-oxide, that favor more compact protein states. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  11. 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.

  12. 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.

  13. Clinical potential of nitric oxide-independent soluble guanylate cyclase activators.

    PubMed

    Doggrell, Sheila A

    2005-09-01

    A major problem with using nitrates in the treatment of ischemic heart disease is that tolerance develops to their vasodilatory actions. YC-1 was used as the lead compound to synthesize further nitric oxide-independent soluble guanylate cyclase activators, including BAY-41-2272 and BAY-41-8543. A nitric oxide and heme-independent activator of soluble guanylate cyclase, BAY-58-2667, was subsequently discovered by high-throughput screening. Tolerance to the vasodilatory actions of BAY-41-8543 and BAY-58-2667 does not develop. Results from animal studies have suggested that these compounds may have potential in the treatment of ischemic heart disease, essential and pulmonary hypertension, congestive heart failure, glomerulonephritis and erectile dysfunction.

  14. p19 detected in the rat retina and pineal gland is a guanylyl cyclase-activating protein (GCAP).

    PubMed

    Dejda, Agnieszka; Matczak, Izabela; Gorczyca, Wojciech A

    2002-01-01

    The Ca(2+)-dependent activation of retina-specific guanylyl cyclase (retGC) is mediated by guanylyl cyclase-activating proteins (GCAPs). Here we report for the first time detection of a 19 kDa protein (p19) with GCAP properties in extracts of rat retina and pineal gland. Both extracts stimulate synthesis of cGMP in rod outer segment (ROS) membranes at low (30 nM) but not at high (1 microM) concentrations of Ca(2+). At low Ca(2+), immunoaffinity purified p19 activates guanylyl cyclase(s) in bovine ROS and rat retinal membranes. Moreover, p19 is recognized by antibodies against bovine GCAP1 and, similarly to other GCAPs, exhibits a Ca(2+)-dependent electrophoretic mobility shift.

  15. Effects of dopamine on adenylyl cyclase activity and amylase secretion in rat parotid tissue.

    PubMed

    Hatta, S; Amemiya, N; Takemura, H; Ohshika, H

    1995-06-01

    Several previous studies have shown that dopamine causes amylase secretion from rat parotid tissue. However, the mechanism of this dopamine action is still unclear. The present study was designed to characterize dopamine action in rat parotid gland tissue by examining the effects of dopamine on cyclic AMP accumulation, adenylyl cyclase activity, and amylase release. Dopamine significantly enhanced accumulation of cyclic AMP in parotid slices and stimulated adenylyl cyclase activity in parotid membrane preparations. It also significantly stimulated amylase release from parotid slices. The stimulatory effects of dopamine on cyclic AMP accumulation, adenylyl cyclase activity, and amylase release were effectively blocked with propranolol, a beta-adrenergic antagonist, but not by either SCH 23390, a preferential D1 antagonist, or butaclamol, a preferential D2 antagonist. No substantial specific binding sites for D1 receptors were detectable by [3H]SCH 23390 binding in parotid membranes. These results suggest that the stimulatory effect of dopamine on amylase secretion in rat parotid tissue is not mediated through specific D1 dopamine receptors but rather through beta-adrenergic receptors.

  16. Comparative study of biological activity of insulins of lower vertebrates in the novel adenylyl cyclase test-system.

    PubMed

    Kuznetsova, L; Shpakov, A; Rusakov, Yu; Plesneva, S; Bondareva, V; Pertseva, M

    2003-11-15

    The biological activity of insulins of lower vertebrates (teleosts-Oncorhynchus gorbuscha, Scorpaena porcus, chondrosteans-Acipenser guldenstaedti and cyclostomates-Lamperta fluviatilis) was studied and compared with that of standard pig insulin. The determination of biological activity was made using the novel adenylyl cyclase (AC) test-system based on the adenylyl cyclase signaling mechanism (ACSM) of insulin action discovered earlier by the authors. The biological activity of insulins was estimated as EC(50), i.e. concentration leading to half-maximal activating effect of the hormone (10(-11)-10(-7) M), in vitro, on adenylyl cyclase in two types of the target tissues: in membrane fractions of the muscles of rat and mollusc Anodonta cygnea. In rat, the efficiency of insulins was found to decrease in the following order: pig insulin>scorpaena insulin>gorbuscha insulin>sturgeon insulin>lamprey insulin. In the mollusc, the order was different: sturgeon insulin>scorpaena insulin>pig insulin>gorbuscha insulin. Lamprey insulin at the same concentrations did not apparently reach the maximal adenylyl cyclase activating effect. The suggestion was made that differences in the biological activity of insulins depend on the hormone structure and a number of indexes characteristic of the adenylyl cyclase test-system in the vertebrate and invertebrate tissues. The proposed adenylyl cyclase test-system is highly sensitive to insulin at physiological concentrations, has good reproduction and is easy to apply.

  17. NO-independent stimulation or activation of soluble guanylyl cyclase during early reperfusion limits infarct size.

    PubMed

    Bice, Justin S; Keim, Yvonne; Stasch, Johannes-Peter; Baxter, Gary F

    2014-02-01

    Guanylyl cyclase-cyclic guanosine monophosphate signalling plays an important role in endogenous cardioprotective signalling. The aim was to assess the potential of direct pharmacological activation and stimulation of soluble guanylyl cyclase, targeting different redox states of the enzyme, to limit myocardial necrosis during early reperfusion. Rat isolated hearts were subjected to reversible left coronary artery occlusion (ischaemia-reperfusion) and infarct size was assessed by the tetrazolium staining technique. Administration during early reperfusion of BAY 41-2272, an NO-independent, haem-dependent stimulator of soluble guanylyl cyclase targeting the reduced state, or BAY 60-2770, an NO-independent, haem-independent activator targeting the oxidized state, significantly limited infarct size. Inhibition of NO synthesis did not abrogate this protection, but exogenous perfusion of NO with BAY 41-2272 produced a synergistic effect. The haem site oxidiser, ODQ abrogated the protection afforded by BAY 41-2272 but potentiated the protection afforded by BAY 60-2770. Targeting both the reduced and oxidized forms of sGC together did not afford additive protection. Targeting either reduced or oxidized forms of sGC during early reperfusion affords cardioprotection, providing support for the concept that direct sGC manipulation at reperfusion has therapeutic potential for the management of acute myocardial infarction.

  18. The soluble guanylyl cyclase activator BAY 60-2770 ameliorates overactive bladder in obese mice.

    PubMed

    Leiria, Luiz O; Silva, Fabio H; Davel, Ana Paula C; Alexandre, Eduardo C; Calixto, Marina C; De Nucci, Gilberto; Mónica, Fabíola Z; Antunes, Edson

    2014-02-01

    Activators of soluble guanylyl cyclase are of potential interest as treatment for cardiovascular diseases but to our knowledge they have never been proposed to treat overactive bladder. We evaluated the effects of the soluble guanylyl cyclase activator BAY 60-2270 on voiding dysfunction and detrusor overactivity in a mouse model of obesity associated overactive bladder. C57BL/6 male mice fed for 10 weeks with standard chow or a high fat diet were treated with 1 mg/kg BAY 60-2770 per day for 2 weeks via gavage. Cystometric evaluations were done and responses to contractile agents in isolated bladders were determined. Obese mice showed an irregular micturition pattern characterized by significant increases in voiding and nonvoiding contractions, which were normalized by BAY 60-2770. Carbachol, KCl and CaCl2 produced concentration dependent contractions in isolated bladder strips, which were markedly greater in obese than in lean mice. BAY 60-2770 normalized bladder contractions in the obese group. A 78% increase in reactive oxygen species generation in the bladder tissue of obese mice was observed, which was unaffected by BAY 60-2770. Treatment with BAY 60-2770 generated a tenfold increase in cyclic guanosine monophosphate in the bladders of obese mice without affecting the nucleotide level in the lean group. Protein expression of the soluble guanylyl cyclase α1 and β1 subunits was decreased 40% in the bladder tissue of obese mice but restored by BAY 60-2770. Two-week BAY 60-2770 therapy increased cyclic guanosine monophosphate and rescued expression of the soluble guanylyl cyclase α1 and β1 subunits in bladder tissue, resulting in great amelioration of bladder dysfunction. Copyright © 2014 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  19. 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.

  20. Selective inhibition of responses to nerve growth factor and of microtubule-associated protein phosphorylation by activators of adenylate cyclase

    PubMed Central

    1986-01-01

    To study the influence of cAMP on cellular responses to nerve growth factor (NGF)