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.

scholarly journals Evidence for regulation of human platelet adenylate cyclase by phosphorylation. Inhibition by ATP and guanosine 5′-[β-thio]diphosphate occur by distinct mechanisms

1991 ◽  
Vol 276 (3) ◽  
pp. 621-630 ◽  
Author(s):  
I A Wadman ◽  
R W Farndale ◽  
B R Martin
Keyword(s):  
Creatine Kinase ◽  
Adenylate Cyclase ◽  
Human Platelet ◽  
Binding Protein ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Phosphoryl Transfer ◽  
Activation Process ◽  
Gtp Binding Protein ◽  
Gtp Binding

1. Incubation of human platelet membranes with guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) causes a time-dependent increase in the activation of adenylate cyclase due to Gs (the stimulatory GTP-binding protein). Forskolin enhances adenylate cyclase activity but does not interfere with the process of activation. The activation follows first-order kinetics in both the presence and the absence of the assay components. 2. ATP in the presence or the absence of an ATP-regenerating system of phosphocreatine and creatine kinase inhibits activation. 3. Hydrolysis of ATP to ADP does not lead to receptor-mediated inhibition of adenylate cyclase acting via Gi (the inhibitory GTP-binding protein). The ADP analogue adenosine 5′-[beta-thio]diphosphate (ADP[S]) does not inhibit the activation process. 4. Phosphocreatine alone inhibits adenylate cyclase activation at concentrations above 1 mM. 5. Inhibition by phosphocreatine is not due to the chelation of free Mg2+ ions. 6. Inhibition by ATP and the other assay components occurs throughout the activation process, decreasing both the rate of activation and the maximum activity obtained. 7. Maximal activation of adenylate cyclase after prolonged incubation with p[NH]ppG slowly reverses in the presence of the assay components. 8. A 10-fold excess of the GDP analogue guanosine 5′-[beta-thio]diphosphate (GDP[S]) over p[NH]ppG inhibits the activation process completely, at all stages of the time course. 9. Preincubations in the presence and absence of ATP, cyclic AMP, phosphocreatine and creatine kinase show equal sensitivity to increasing GDP[S] concentration. These data show that the inhibition observed in the presence of ATP is not due to endogenous or contaminating guanine nucleotides, and suggest that phosphoryl transfer may regulate adenylate cyclase activity.

scholarly journals Role of pertussis toxin-sensitive guanosine triphosphate-binding proteins in the response of erythroblasts to erythropoietin

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 486-492 ◽  
Author(s):  
BA Miller ◽  
K Foster ◽  
JD Robishaw ◽  
CF Whitfield ◽  
L Bell ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Pertussis Toxin ◽  
Binding Proteins ◽  
Binding Protein ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Guanosine Triphosphate ◽  
Gtp Binding Protein ◽  
Gtp Binding

Abstract Human progenitor-derived erythroblasts have been recently shown to respond to erythropoietin (Epo) with an increase in intracellular free calcium concentration [Cac]. To explore the role of guanosine triphosphate (GTP)-binding proteins in mediating the rise in [Cac], single day 10 erythroid burst forming unit (BFU-E)-derived erythroblasts loaded with Fura-2 were pretreated with pertussis toxin (PT), stimulated with Epo, and [Cac] measured over 18 minutes with fluorescence microscopy coupled to digital video imaging. The [Cac] increase in day 10 erythroblasts stimulated with Epo was blocked by pretreatment with PT in a dose-dependent manner but not by heat- inactivated PT. These observations provided strong evidence that a PT- sensitive GTP-binding protein is involved. To further characterize the GTP-binding protein, day 10 erythroblast membrane preparations were solubilized, electrophoresed, and immunoblotted with antibodies specific for the known PT-sensitive G-protein subunits: the three subtypes of Gia (1,2, and 3) and Goa, Gia1 or Gia3 and Gia2 were identified but no Goa was found. To examine the influence of Epo on adenylate cyclase activity, day 10 erythroblasts were initially treated with Epo, isolated membrane preparations made, and cyclic adenosine monophosphate (cAMP) production by adenylate cyclase in membrane preparations in the presence of theophylline measured. Epo did not inhibit but significantly stimulated adenylate cyclase activity. However, the mechanism of increase of [Cac] appears to be independent of adenylate cyclase stimulation because treatment of erythroblasts with the cell-permeant dibutyryl cAMP failed to increase [Cac]. In summary, pertussis toxin blocks the increase in [Cac] in erythroblasts after Epo stimulation suggesting that this response is mediated through a pertussis toxin-sensitive GTP-binding protein. Candidate PT-sensitive GTP-binding proteins identified on day 10 erythroblasts were Gia 1, 2, or 3, but not Goa.

A proper transmembrane Ca2+ gradient is essential for the stimulation of adenylate cyclase activity by stimulatory GTP-binding protein

1994 ◽  
Vol 14 (4) ◽  
pp. 179-187 ◽  
Author(s):  
Youguo Huang ◽  
Gaofeng Fan ◽  
Fuyu Yang
Keyword(s):  
Adenylate Cyclase ◽  
Conformational Changes ◽  
Physiological Condition ◽  
Fluorescence Probe ◽  
Binding Protein ◽  
Bovine Brain ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Gtp Binding Protein ◽  
Gtp Binding

Stimulatory GTP-binding Protein (Gs) and adenylate cyclase prepared from bovine brain cortices were co-reconstituted into asolectin vesicles with or without 1000-fold transmembrane Ca2+ gradient. The results showed that both basal activity and Gs-stimulated activity of adenylate cyclase were highest in proteoliposomes with a transmembrane Ca2+ gradient similar to physiological condition (1 μM Ca2+ outside and 1 mM Ca2+ inside) and lowest when the transmembrane Ca2+ gradient was in the inverse direction. Such a difference could be diminished following dissipation of the transmembrane Ca2+ gradient by A23187. Comparable conformational changes of Gs in proteoliposomes were also observed when Gs was labeled with the fluorescence probe, acrylodan. These results may indicate that a proper transmembrane Ca2+ gradient is essential not only for higher adenylate cyclase activity but also for its stimulation by Gs.

scholarly journals Guanine nucleotide-independent inhibition of adenylate cyclase by a stimulatory hormone

1988 ◽  
Vol 254 (1) ◽  
pp. 27-31 ◽  
Author(s):  
C Greiner ◽  
K H Jakobs
Keyword(s):  
Adenylate Cyclase ◽  
Binding Protein ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Lag Phase ◽  
Hybrid Cells ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Dual Action ◽  
Activity State

Stimulation of basal adenylate cyclase activity in membranes of neuroblastoma x glioma hybrid cells by prostaglandin E1 (PGE1) is half-maximal and maximal (about 8-fold) at 0.1 and 10 microM respectively. This hormonal effect requires GTP, being maximally effective at 10 microM. However, at the same concentrations that stimulate adenylate cyclase in the presence of GTP, PGE1 inhibited basal adenylate cyclase activity when studied in the absence of GTP, by maximally 60%. A similar dual action of PGE1 was observed with the forskolin-stimulated adenylate cyclase, although the potency of PGE1 in both stimulating and inhibiting adenylate cyclase was increased and the extent of stimulation and inhibition of the enzyme by PGE1 was decreased by the presence of forskolin. The inhibition of forskolin-stimulated adenylate cyclase by PGE1 occurred without apparent lag phase and was reversed by GTP and its analogue guanosine 5′-[gamma-thio]triphosphate at low concentrations. Treatment of neuroblastoma x glioma hybrid cells or membranes with agents known to eliminate the function of the inhibitory GTP-binding protein were without effect on PGE1-induced inhibition of adenylate cyclase. The data suggest that stimulatory hormone agonist, apparently by activating one receptor type, can cause both stimulation and inhibition of adenylate cyclase, and that the final result depends only on the activity state of the stimulatory GTP-binding protein, Gs. Possible mechanisms responsible for the observed adenylate cyclase inhibition by the stimulatory hormone PGE1 are discussed.

Mechanism of Li inhibition of vasopressin-sensitive adenylate cyclase in cultured renal epithelial cells

1988 ◽  
Vol 255 (5) ◽  
pp. F995-F1002 ◽  
Author(s):  
H. Goldberg ◽  
P. Clayman ◽  
K. Skorecki
Keyword(s):  
Adenylate Cyclase ◽  
Binding Protein ◽  
Inhibitory Effect ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Ionophore A23187 ◽  
Camp Production ◽  
Gtp Binding Protein ◽  
Intact Cells ◽  
Gtp Binding

We investigated the mechanism for lithium-induced inhibition of vasopressin-stimulated adensoine 3',5'-cyclic monophosphate (cAMP) production in the renal epithelial cell line LLC-PK1. In LLC-PK1 membranes lithium caused direct inhibition of hormone-stimulated adenylate cyclase activity by competing with magnesium. Fifty percent inhibition occurred at 20 mM lithium. The maximum transport activity (Vmax) but not the activation constant (Ka) for activation by vasopressin was altered. Activation by GTP and its nonhydrolyzable analogues was also inhibited by lithium. Furthermore, kinetic studies revealed that the lag phase in the activation of adenylate cyclase by 5'-guanylimi-dotriphosphate [Gpp(NH)p] was prolonged from 1 to 3 min, suggesting an effect of lithium on magnesium-dependent activation of the stimulatory GTP binding protein Gs. The function of the corresponding inhibitory GTP-binding protein Gi, as assessed by GTP inhibition of vasopressin-stimulated adenylate cyclase activity in the presence and absence of pertussis toxin pretreatment, was unaffected. Intact LLC-PK1 cells incubated in 10 mM lithium (approximate urinary concentration in lithium-treated patients) attained an intracellular lithium concentration of 17 mM, which led to a 40% reduction in cAMP formation. Magnesium loading of intact cells with the ionophore A23187 reversed the inhibitory effect of lithium. It is concluded that lithium directly inhibits the activation of vasopressin-sensitive adenylate cyclase in renal epithelia by competing with magnesium for activation of Gs. This direct effect on Gs activation accounts for the inhibitory effect of lithium on cAMP production in the intact cell.

GTP-binding proteins and adenylate cyclase activity in v-Ki-ras transformed NIH/3T3 fibroblast cells

1988 ◽  
Vol 153 (3) ◽  
pp. 1214-1222 ◽  
Author(s):  
Lisa Konishi-Imamura ◽  
Michihisa Tohda ◽  
Makoto Noda ◽  
Hirofumi Nakano ◽  
Yasuyuki Nomura
Keyword(s):  
Adenylate Cyclase ◽  
Binding Proteins ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Fibroblast Cells ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Nih 3T3
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