The hysteretic effect of Gpp(NH)p on adenylate cyclase is not altered by Mg2+ in adipocyte membranes of ob/ob mice

1986 ◽  
Vol 64 (9) ◽  
pp. 855-863 ◽  
Author(s):  
Nicole Bégin-Heick
Keyword(s):  
Adipose Tissue ◽  
Steady State ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Incubation Medium ◽  
Time Course ◽  
Inhibitory Effect ◽  
Lag Phase ◽  
Guanine Nucleotide ◽  
Hysteretic Effect

We have established previously that the regulation of adenylate cyclase is abnormal in adipose tissue membranes of ob/ob mice. To help establish the nature of the defect, we studied the time course of guanine nucleotide activation and inhibition of adenylate cyclase. The activation of adenylate cyclase by Gpp(NH)p in adipocyte membranes of normal (+/+) and ob/ob mice proceeds with a lag phase. In +/+ membranes, this lag could be shortented by increasing the concentration of Mg2+ in the incubation medium or by pretreatment of the membranes with cholera toxin, and it could be abolished by isoproterenol in combination with 4 mM MgCl2. In contrast, in the ob/ob membranes, only pretreatment with cholera toxin was effective in shortening the lag phase. These results indicate an impediment in the activation of adenylate cyclase in ob/ob membranes. In the +/+ membranes, Gpp(NH)p inhibited forskolin-stimulated adenylate cyclase, following a short lag phase, producing lower steady-state velocities than those seen with forskolin alone. The inhibitory effect of Gpp(NH)p on forskolin-stimulated activity was abolished by pertussis but not by cholera toxin treatment. In the ob/ob membranes, neither Gpp(NH)p nor pertussis treatment had any effect on the steady-state velocity of the forskolin-stimulated activity. These data have been interpreted as meaning that an anomaly in Ni rather than in Ns is likely to be responsible for the impairment of adenylate cyclase activity in the membranes of the ob/ob mouse.

Determining steps in the regulatory GTPase cycle of rat pancreatic adenylate cyclase

1981 ◽  
Vol 296 (1080) ◽  
pp. 139-150 ◽  
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Time Course ◽  
Plasma Membranes ◽  
Fold Increase ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Control Value ◽  
Activation Rate ◽  
Gtpase Cycle

The time course of activation and deactivation and the degree of activation at steady state [ E a ] / [ E tot ] of adenylate cyclase, in semi-purified rat pancreatic plasma membranes, were compatible with a simple two-state model with three rate constants, so that [ E a ] / [ E tot ] = k +1 /( k +1 + k 2 + k -1 ). The hormone CCK-8 increased k +1 with GTP in a dose-dependent manner, from 0.2 to 10.9 min -1 ; k -1 increased from 0.01 to 0.3 min -1 , i.e. in proportion, but k 2 was unaltered at 7 min -1 , so that [ E a ] / [ E tot ] increased 15-fold, from 4 to 61%. A similar activation was obtained after cholera toxin pretreatment but by a different mechanism. The toxin pretreatment exerted a major inhibitory effect on the value of k 2 and on the corresponding GTPase activity. A pretreatment at the high cholera toxin concentration (30 pg/ml) exerted two additional effects that became evident when p[NH]ppG rather than GTP was used as activating nucleotide: ( a ) a relatively large increase in k -1 from an unmeasurably low control value to 0.3 min -1 , and ( b ) a four-fold increase in the p[NH]ppG activation rate, k +1. This contrasted with the action of CCK-8, which increased k -1 and k +1 in proportion.

PGE2, forskolin, and cholera toxin interactions in rabbit cortical collecting tubule

1986 ◽  
Vol 250 (1) ◽  
pp. F127-F135 ◽  
Author(s):  
S. P. Nadler ◽  
S. C. Hebert ◽  
B. M. Brenner
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Regulatory Protein ◽  
Inhibitory Effect ◽  
Lag Phase ◽  
Collecting Tubule ◽  
Dose Dependent Fashion ◽  
Pg E2 ◽  
Rabbit Cortical Collecting Tubule ◽  
Cortical Collecting Tubule

To define further the mechanism whereby prostaglandin (PG) E2 inhibits the hydroosmotic response to ADH, we studied the interactions of PGE2 with ADH and two nonhormonal activators of adenylate cyclase, forskolin and cholera toxin, in the isolated perfused rabbit cortical collecting tubule. Forskolin increased hydraulic conductivity (LP) in a dose-dependent fashion and to a degree comparable with ADH-stimulated LP. Forskolin also augmented maximal ADH-stimulated LP, from 135 +/- 15 (SE) to 174 +/- 7 . 10(-7) cm . s-1 . atm-1. Following a 45-min lag phase, 10(-9) M cholera toxin at 37 degrees C increased LP to 107 +/- 12 . 10(-7) cm . s-1 . atm-1, a response that was stable with time. In paired studies at both 25 and 37 degrees C, PGE2 reversibly inhibited ADH-stimulated LP by 45 and 47%, respectively. However, the same protocols with PGE2 and forskolin failed to reveal any inhibitory effect of PGE2 on forskolin-stimulated LP. PGE2 reversibly inhibited cholera toxin-stimulated LP, from 124 +/- 15 to 100 +/- 15 . 10(-7) cm . s-1 . atm-1. These results support the view that PGE2 inhibits ADH-stimulated LP by inhibiting the synthesis of cAMP and suggest that this inhibition occurs at a functional site at or distal to the nucleotide regulatory protein of adenylate cyclase.

Uncoupling of Lipolysis from Cyclic AMP by Procaine: a Tool for Studying the Mechanism of Action of Antilipolytic Agents

1975 ◽  
Vol 53 (4) ◽  
pp. 603-609 ◽  
Author(s):  
Mario D'Costa ◽  
Aubie Angel
Keyword(s):  
Adipose Tissue ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Mechanism Of Action ◽  
Initial Rate ◽  
Direct Action ◽  
Inhibitory Effect ◽  
Glycerol Release ◽  
Cyclic Amp Accumulation ◽  
Low K

The initial rate of net glycerol release in norepinephrine-stimulated adipose tissue fragments was inhibited (40–78%) by procaine–HCl (1–5 mM), whereas basal (unstimulated) lipolysis was unaffected. A dose-related inhibition of norepinephrine-induced lipolysis by procaine–HCl (0.1–1 mM) also occurred in adipocytes. Procaine-induced antilipolysis was associated with an augmented rather than a reduced hormone-stimulated increment in intracellular cyclic AMP. The dissociation of lipolysis from cyclic AMP accumulation has been termed the uncoupling effect of procaine. This effect of procaine was employed to define the precise mechanism of action of the antilipolytic drug clofibrate (Atromid-S®) which inhibits lipolysis by reducing cyclic AMP. A reduction in cyclic AMP by clofibrate was demonstrated in norepinephrine-stimulated cells exposed to procaine (uncoupled system). Thus, the inhibitory effect of clofibrate on cyclic AMP could not be attributed to accumulation of products of lipolysis. Because neither procaine–HCl nor clofibrate had any effect on the low Km 3′:5′-cyclic-AMP phosphodiesterase (EC 3.1.4.17) activity in hormone stimulated cells, the clofibrate-induced reduction in cyclic AMP was attributed to its direct action on adipocyte adenylate cyclase.

scholarly journals Cytochemical studies of myocardial adenylate cyclase after its activation and inhibition.

1984 ◽  
Vol 32 (1) ◽  
pp. 105-113 ◽  
Author(s):  
J Slezak ◽  
S A Geller
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Adenylate Cyclase ◽  
Adenosine Deaminase ◽  
Incubation Medium ◽  
Calcium Release ◽  
Inhibitory Effect ◽  
Alkaline Phosphatases ◽  
Similar Role ◽  
Adenosine Triphosphatases ◽  
Junctional Sarcoplasmic Reticulum

Incubation medium, as previously described (J Histochem Cytochem 27:774, 1979), was used to demonstrate the presence of adenylate cyclase (AC) in myocardium. NaF and ouabain were used to inhibit adenosine triphosphatases (ATP) and NaF and isoproterenol were used as activators of AC. The inhibitory effect of adenosine on AC was blocked by the addition of adenosine deaminase. The addition of tetramisol blocked the influence of the alkaline phosphatases on adenylyl imidodiphosphate hydrolysis. The use of these substances resulted in specific precipitation localized in junctional sarcoplasmic reticulum and sarcolemma. The reaction product was dramatically intensified after activation of AC by NaF or isoproterenol. Preincubation in 10-100 mM of propranolol, for 30 min, blocked AC stimulation by isoproterenol and prevented the appearance of the specific precipitate. The localization of specific precipitate in junctional sarcoplasmic reticulum and subsarcolemmal cisternae corresponds to the localization of Na+, K+ ATPase and may reflect the similar role that AC and Na+, K+ ATPase play in calcium release from sarcoplasmic reticulum of internal and peripheral couplings.

scholarly journals Interactions of cholera toxin with isolated hepatocytes. Effects of low pH, chloroquine and monensin on toxin internalization, processing and action

1988 ◽  
Vol 253 (3) ◽  
pp. 735-743 ◽  
Author(s):  
M Janicot ◽  
J P Clot ◽  
B Desbuquois
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Acidic Medium ◽  
Isolated Hepatocytes ◽  
Surface Generation ◽  
Lag Phase ◽  
Single Class ◽  
Endosomal Membrane ◽  
A Subunit

The major steps in cholera-toxin action, i.e. binding, internalization, generation of A1 peptide and activation of adenylate cyclase, were examined in isolated hepatocytes. The binding of toxin involves a single class of high-affinity sites (KD congruent to 0.1 nM; Bmax. congruent to 10(7) sites/cell). At 37 degrees C, cell-associated toxin is progressively internalized, as judged by the loss of its accessibility to antibodies against whole toxin, A and B subunits (about 50, 75 and 30% of initially bound toxin after 40 min respectively). Two distinct pathways are involved in this process: endocytosis of the whole toxin, and selective penetration of the A subunit into the plasma membrane. Exposure of hepatocytes to an acidic medium (pH 5) results in a rapid and marked disappearance of the A subunit from the cell surface. Generation of A1 peptide and activation of adenylate cyclase by the toxin occur after a lag phase (10 min at 37 degrees C), and increase with time in a parallel manner up to 2-3% A1 peptide generated; they are unaffected by exposure of cells to an acidic medium. Chloroquine and monensin, which elevate the pH in acidic organelles, inhibit by 2-4-fold both the generation of A1 peptide and the activation of adenylate cyclase. Unexpectedly, these drugs also inhibit the internalization of the toxin. These results suggest that an acidic pH facilitates the penetration of A subunit into the plasma membrane and presumably the endosomal membrane as well, and that endocytosis of cholera toxin is required for generation of A1 peptide and activation of adenylate cyclase.

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.

scholarly journals Activation of adenylate cyclase in bovine corpus-luteum membranes by human choriogonadotropin, guanine nucleotides and NaF

1981 ◽  
Vol 198 (3) ◽  
pp. 631-638 ◽  
Author(s):  
Nicholas B. Lydon ◽  
John L. Young ◽  
David A. Stansfield
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Guanine Nucleotides ◽  
Lag Phase ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Human Choriogonadotropin ◽  
Activated State ◽  
Cyclic Amp Synthesis

1. Preincubation of luteal membranes with human choriogonadotropin results in the formation of an activated state of adenylate cyclase which is not reversed by washing and which is limited only by the absence of guanine nucleotides, whereas preincubation with GTP yields only a partially activated adenylate cyclase which requires the presence of both GTP and human choriogonadotropin during assay to demonstrate maximal activity. 2. Preincubation of luteal membranes with GTP and human choriogonadotropin does not lead to a synergistic increase in wash-resistant activity. 3. Luteal membranes that had been preincubated with GTP and hormone exhibited a decreasing rate of cyclic AMP synthesis during the adenylate cyclase assay incubation; addition of GTP during the assay incubation reversed the decrease. 4. Membranes that had been preincubated in the absence of guanine nucleotide and hormone showed a ‘burst’ phase of cyclic AMP synthesis when GTP was present in the assay incubation and a ‘lag’ phase with p[NH]ppG (guanosine 5′-[β,γ-imido]triphosphate) present in the assay. The presence of human choriogonadotropin with either nucleotide in the assay incubation eliminated the curvatures in plots observed with guanine nucleotides alone. 5. Luteal adenylate cyclase was persistently activated by preincubation with p[NH]ppG alone or in combination with human choriogonadotropin; the activation caused by p[NH]ppG alone was still increasing after 70min of preincubation, whereas that caused by p[NH]ppG in the presence of hormone was essentially complete within 10min of preincubation. 6. Luteal adenylate cyclase that had been partially preactivated by preincubation with p[NH]ppG was slightly increased in activity by the inclusion of further p[NH]ppG in the adenylate cyclase assay incubation, but more so with p[NH]ppG and hormone. Human choriogonadotropin alone caused no further increase in the activity of the partially stimulated preparation unless p[NH]ppG was also added to the assay incubation. 7. GTP decreased the activity of adenylate cyclase in membranes that had been partially preactivated in the presence of p[NH]ppG; the decrease in activity was greater when GTP and hormone were present simultaneously in the assay. 8. The results indicate that stable activation states of adenylate cyclase can be induced by preincubation of luteal membranes in vitro with human choriogonadotropin or p[NH]ppG, and that in the presence of p[NH]ppG the hormone may accelerate events subsequent to guanine nucleotide binding. Stable activation of luteal adenylate cyclase by prior exposure to GTP is not achieved. The involvement of GTPase activity and of hormone-promoted guanine nucleotide exchange in the modulation of luteal adenylate cyclase activity is discussed.

scholarly journals Studies on the time course and rate-limiting steps in the activation of adenylate cyclase in rat liver by cholera toxin

1978 ◽  
Vol 173 (1) ◽  
pp. 59-64 ◽  
Author(s):  
J Fischer ◽  
T R Kohler ◽  
L G Lipson ◽  
J Flores ◽  
P A Witkum ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Rat Liver ◽  
Time Course ◽  
Intact Cell ◽  
Intact Cells ◽  
Maximal Stimulation ◽  
Rate Limiting

Cholera toxin stimulates adenylate cyclase in rat liver after intravenous injection. The stimulation follows a short latent period of 10min, and maximum stimulation was attained at 120min. Half-maximal stimulation was achieved at 35min. In contrast with this lengthy time course in the intact cell, adenylate cyclase in broken-cell preparations of rat liver in vitro were maximally stimulated by cholera toxin (in the presence of NAD+) in 20min with half-maximal stimulation in 8min. Binding of cholera toxin to cell membranes by the B subunits is followed by translocation of the A subunit into the cell or cell membrane, and separation of the A1 polypeptide chain from the A2 chain by disulphide-bond reduction, and finally activation of adenylate cyclase by the A1 chain and NAD+. As the binding of cholera toxin is rapid, two possible rate-limiting steps could be the determinants of the long time course of action. These are translocation of the A1 chain from the outside of the cell membrane to its site of action (this includes the time required for separation from the whole toxin) or the availability of NAD+ for activation. When NAD+ concentrations in rat liver were elevated 4-fold, by the administration of nicotinamide, no change in the rate of activation of adenylate cyclase by cholera toxin was observed. Thus the intracellular concentration of NAD+ is not rate-limiting and the major rate-limiting determinant in intact cells must be between the time of toxin binding to the cell membrane and the appearance of subunit A1 at the enzyme site.

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