scholarly journals Gsα is a substrate for mono(ADP-ribosyl)transferase of NG108-15 cells. ADP-ribosylation regulates Gsα activity and abundance

1992 ◽  
Vol 288 (1) ◽  
pp. 331-336 ◽  
Author(s):  
L E Donnelly ◽  
R S Boyd ◽  
J MacDermot
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Cell Membranes ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Major Protein ◽  
Adp Ribosylation ◽  
Short Term Exposure ◽  
Significant Difference ◽  
Gs Alpha

NG108-15 neuroblastoma x glioma somatic hybrid cells were permeabilized in the presence of [32P]NAD+ and then cultured for 18 h. Resolution of the cell proteins on polyacrylamide gels revealed [32P]ADP-ribosylation of five major protein species with molecular mass values of 52 kDa, 44 kDa, 35 kDa, 30 kDa and 25 kDa. A similar pattern of labelling was also seen when NG108-15 cell membranes were incubated with [32P]NAD+ and hydrolysis of the product revealed mono(ADP-ribosyl)ation. Immunoprecipitation of these products with anti-Gs alpha antiserum revealed a single band identical to cholera toxin substrate. Culture of [32P]NAD(+)-loaded cells for 18 h in the presence of 50 mM-nicotinamide inhibited the eukaryotic mono(ADP-ribosyl)transferase activity. Inhibition of the eukaryotic enzyme was also accompanied by an increase in the abundance of Gs alpha, whether measured by Western blotting with anti-Gs alpha antibody (two separate antisera) or by cholera toxin-dependent [32P]ADP-ribosylation. There was no accompanying change in the abundance of G beta. The increase in Gs alpha abundance in nicotinamide-treated NG108-15 cells was accompanied by a 2-fold increase in basal adenylate cyclase activity (measured in the presence of GTP), and by a smaller but significant increase in iloprost-dependent activation of adenylate cyclase. Receptor number or affinity was not affected by nicotinamide, since this treatment did not alter the binding parameters of [3H]iloprost to NG108-15 cell membranes. Short-term exposure of cells to nicotinamide for 1 h revealed no significant difference in either basal or agonist-stimulated adenylate cyclase activity. These results reveal that mono(ADP-ribosyl)ation of Gs alpha by eukaryotic ADP-ribosyltransferase modifies the abundance and activity of Gs alpha in NG108-15 cells, and hence may play a role in the hormonal regulation of cell function.

Modulation of Gs activity by phorbol myristate acetate in rat hepatocytes

1991 ◽  
Vol 260 (2) ◽  
pp. C259-C265 ◽  
Author(s):  
S. M. Hernandez-Sotomayor ◽  
M. Macias-Silva ◽  
C. C. Malbon ◽  
J. A. Garcia-Sainz
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Rat Hepatocytes ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Heterologous Desensitization ◽  
G Protein Alpha Subunit ◽  
Activity Catalyst ◽  
Gs Alpha ◽  
Mouse Lymphoma

Activation of protein kinase C promotes heterologous desensitization of hepatic adenylate cyclase. The basis for this desensitization was explored by use of a strategy with several independent approaches. Although not influencing the amount of forskolin-stimulated adenylate cyclase activity (catalyst), treatment with phorbol 12-myristate 13-acetate (PMA) decreased adenylate cyclase activation in response to either sodium fluoride or guanylyl imidodiphosphate [Gpp(NH)p]. Adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in cholera toxin-treated hepatocytes and both the basal and GTP-stimulated adenylate cyclase activity of membranes from toxin-treated cells displayed a marked reduction in response to PMA. The ability of cholate extracts of hepatocyte membranes to reconstitute beta-adrenergic-stimulated adenylate cyclase activity of membrane of S49 mouse lymphoma cyc- cells was reduced by treatment with PMA. Cholera toxin-catalyzed labeling of Gs alpha-subunits was likewise diminished by phorbol ester treatment. Immunoblots of membranes from control or PMA-treated hepatocytes showed no difference in the amount of Gs alpha. Immunoprecipitation studies failed to detect phosphorylation of this G protein alpha-subunit. The data demonstrate that PMA induces an alteration in the functional status of Gs without altering the amount of this transmembrane signaling element. The alteration in Gs function may play a significant role in heterologous desensitization.

scholarly journals Bradykinin-dependent activation of adenylate cyclase activity and cyclic AMP accumulation in tracheal smooth muscle occurs via protein kinase C-dependent and -independent pathways

1994 ◽  
Vol 297 (1) ◽  
pp. 233-239 ◽  
Author(s):  
P A Stevens ◽  
S Pyne ◽  
M Grady ◽  
N J Pyne
Keyword(s):  
Smooth Muscle ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Phospholipase D ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Tracheal Smooth Muscle ◽  
Constitutive Activation ◽  
Cyclic Amp Accumulation

Treatment of cultured tracheal smooth-muscle cells (TSM) with phorbol 12-myristate 13-acetate (PMA) (100 nM) or bradykinin (100 nM) elicited enhanced basal and guanosine 5′-[beta gamma-imido]-triphosphate-stimulated adenylate cyclase activities in subsequently isolated membranes. Combined stimulation of cells was non-additive, indicating that both agents activate adenylate cyclase via similar routes. Both PMA (100 nM) and bradykinin (100 nM) allowed the alpha subunit of Gs to act as a more favourable substrate for its cholera-toxin-catalysed ADP-ribosylation in vitro. PMA was without effect on intracellular cyclic AMP in control cells. However, constitutive activation of Gs by treatment in vivo with cholera toxin (0.5 ng/ml, 18 h) sensitized the cells to PMA stimulation, resulting in a concentration-dependent increase in intracellular cyclic AMP accumulation (EC50 = 7.3 +/- 2.5 nM, n = 5). Bradykinin also elicited a concentration-dependent increase in intracellular cyclic AMP (EC50 = 63.3 +/- 14.5 nM, n = 3). Constitutive activation of Gs resulted in an increased maximal response (10-fold) and potency (EC50 = 6.17 +/- 1.6 nM, n = 3) to bradykinin. This response was not affected by the B2-receptor antagonist, NPC567 [which selectively blocks bradykinin-stimulated phospholipase C (PLC), with minor activity against phospholipase D (PLD) activity]. Des-Arg9-bradykinin (a B1-receptor agonist) was without activity. These results suggest that the receptor sub-type capable of activating PLD may also be stimulatory for cyclic AMP accumulation. Furthermore, pre-treatment of the cells with butan-l-ol (0.3%, v/v), which traps phosphatidate derived from PLD reactions, blocked the bradykinin-stimulated increase in intracellular cyclic AMP. These studies suggest that there may be a causal link between PLD-derived phosphatidate and the positive modulation of adenylate cyclase activity. In support of this, the concentration-dependence for bradykinin-stimulated adenylate cyclase activity was identical with that of bradykinin-stimulated phospholipase D activity (EC50 = 5 nM). Bradykinin, but not PMA, was also capable of eliciting the inhibition of cyclic AMP phosphodiesterase activity in TSM cells (EC50 > 100 nM) via an unidentified mechanism. These studies indicate that cross-regulation between the cyclic AMP pathway and phospholipid-derived second messengers in TSM cells does not occur as a consequence of PLC-catalysed PtdIns(4,5)P2 hydrolysis, but may involve, in part, PLD-catalysed phosphatidylcholine hydrolysis.

scholarly journals Insulin and vasopressin elicit inhibition of cholera-toxin-stimulated adenylate cyclase activity in both hepatocytes and the P9 immortalized hepatocyte cell line through an action involving protein kinase C

1995 ◽  
Vol 312 (3) ◽  
pp. 769-774 ◽  
Author(s):  
L Zeng ◽  
M D Houslay
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Cell Line ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
3T3 Cells ◽  
Kinase C ◽  
Nih 3T3

Incubation of hepatocytes or the SV40-DNA-immortalized hepatocyte P9 cell line with cholera toxin led to a time-dependent activation of adenylate cyclase activity, which occurred after a defined lag period. When added together with cholera toxin, each of the hormones insulin and vasopressin was capable of attenuating the maximum stimulatory effect achieved by cholera toxin over a period of 60 min through a process which could be blocked by the compounds staurosporine and chelerythrine. Attenuating effects on cholera-toxin-stimulated adenylate cyclase activity could also be elicited by using either the protein kinase C (PKC)-stimulating phorbol ester PMA (phorbol 12-myristate 13-acetate) or the protein phosphatase inhibitor okadaic acid. Alkaline phosphatase treatment of membranes reversed the inhibitory effect of PMA. Cholera toxin also stimulated the adenylate cyclase activity of intact CHO (Chinese-hamster ovary) and NIH-3T3 cells, but this activity was insensitive to the addition of PMA. Overexpression of various PKC isoforms in CHO cell lines did not confer sensitivity to inhibition by PMA upon cholera-toxin-stimulated adenylate cyclase activity. Rather, overexpression of the gamma isoform of PKC allowed PMA to stimulate adenylate cyclase activity in CHO cells. It is suggested that the PKC-mediated phosphorylation of a membrane protein attenuates cholera-toxin-stimulated adenylate cyclase activity in hepatocytes and P9 cells. The cellular selectivity of such an action may be due to the target for this inhibitory action of PKC being a particular isoform of adenylate cyclase which provides the major activity in hepatocytes and P9 cells, but not in either CHO or NIH-3T3 cells.

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

1993 ◽  
Vol 265 (4) ◽  
pp. G686-G698 ◽  
Author(s):  
B. S. Dixon ◽  
E. Sutherland ◽  
A. Alexander ◽  
D. Nibel ◽  
F. R. Simon
Keyword(s):  
Adenylate Cyclase ◽  
Apical Membrane ◽  
Bile Duct Ligation ◽  
Basolateral Membrane ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Gtp Binding ◽  
Duct Ligation ◽  
Apical Membranes ◽  
Gs Alpha

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.

Tannin inhibits adenylate cyclase in airway epithelial cells

1995 ◽  
Vol 268 (5) ◽  
pp. L851-L855
Author(s):  
M. M. Cloutier ◽  
L. Guernsey
Keyword(s):  
Epithelial Cells ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Airway Epithelial Cells ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Airway Epithelial ◽  
Camp Production ◽  
Dose Dependent ◽  
In Cells

Tannin, isolated from cotton bracts extract and implicated in the pathogenesis of byssinosis, inhibits adenosine 3',5'-cyclic monophosphate (cAMP) production and Cl- secretion in bovine airway epithelial cells in part by inhibiting adrenergic receptor binding. The purpose of this study was to determine whether tannin affected other parts of the adrenergic-cAMP signal transduction pathway by examining the effect of tannin on guanosine 5'-triphosphate (GTP)-regulatory pathways (G proteins) and on adenylate cyclase activity. cAMP production in confluent airway epithelial cells was measured in the presence of cholera toxin (100 micrograms/ml), an activator of GS proteins, and forskolin (0.1-1,000 microM), a direct activator of adenylate cyclase. Cholera toxin stimulated cAMP production; this response, however, was inhibited in cells pretreated with 50 micrograms/ml tannin. Forskolin (100 microM) stimulated cAMP production 13-fold above baseline values. Tannin pretreatment inhibited the stimulatory effect of forskolin on cAMP release in a dose-dependent manner with a tannin concentration causing 50% inhibition of 7.5 micrograms/ml. The stimulatory effect of forskolin on cAMP release was completely inhibited in cells pretreated with 50 micrograms/ml tannin. The inhibition was reversible 3 h after removal of tannin from the solution. Tannin also inhibited forskolin-stimulated adenylate cyclase activity in a dose-dependent, noncompetitive manner. We conclude that forskolin and cholera toxin stimulate cAMP production in airway epithelial cells and that tannin inhibits the production of cAMP in airway epithelial cells by a direct effect on adenylate cyclase activity.

scholarly journals Human platelets are defective in processing of cholera toxin

1983 ◽  
Vol 212 (3) ◽  
pp. 669-678 ◽  
Author(s):  
R J Hughes ◽  
P A Insel
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Mammalian Cells ◽  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity

Cholera toxin is unable to elevate cyclic AMP levels in intact human platelets despite being very efficacious in this respect in other mammalian cells; in the presence of 0.5 mM-isobutylmethylxanthine, we found that 3-6nM-cholera toxin over 3h at 37 degrees C elevated platelet cyclic AMP from 33 +/- 13 to 39 +/- 12pmol/mg of protein (means +/- S.D.; n = 12). We have investigated the basis for this lack of response. 125I-labelled cholera toxin bound to platelets both saturably and with high affinity (Kd congruent to 60pM; Bmax. congruent to 50fmol/mg of protein). Incubation of platelets with the putative cholera toxin receptor monosialoganglioside GM1 enhanced 125I-labelled cholera toxin binding at least 40-fold but facilitated only a minimal (less than or equal to 3-fold) elevation of platelet cyclic AMP levels. In contrast, dithiothreitol-activated cholera toxin markedly stimulated adenylate cyclase activity in platelet membranes. Platelet cytosol both enhanced stimulation of adenylate cyclase activity by activated cholera toxin (A1 subunit) and supported stimulation by the A1-A2 subunit of cholera toxin. Neither GTP nor NAD+, both necessary for response to cholera toxin, was lacking in intact platelets. However, we found that platelets were unable to cleave cholera toxin to the active A1 subunit (as assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis). By contrast, murine S49 lymphoma cells were able to generate the A1 subunit with a time course that closely resembled the kinetics of toxin-mediated cyclic AMP accumulation in these cells. Thus we conclude that human platelets are defective in their ability to process surface-bound cholera toxin. These results indicate that binding of cholera toxin to surface receptors is necessary, but not sufficient, for expression of the toxin effect and the generation of the A1 subunit of the toxin may be rate-limiting for expression of cholera toxin response.

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