scholarly journals Modulation of the response of bovine adrenocortical adenylate cyclase to corticotropin

1977 ◽  
Vol 168 (2) ◽  
pp. 277-282 ◽  
Author(s):  
P Glynn ◽  
D M F Cooper ◽  
D Schulster
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Guanine Nucleotide ◽  
Half Maximum ◽  
Basic Parameters ◽  
High Concentrations ◽  
Plasma Membrane Preparation ◽  
Stimulation Of

An assessment was made of some of the basic parameters responsible for the modulation of adenylate cyclase activity in a bovine adrenocortical plasma-membrane preparation. When determined at 0.1 mM-ATP, basal adenylate cyclase activity increased with increasing MgCl2 concentrations, whereas in the presence of corticotropin activity was essentially maximal at 10mM-MgCl2; high concentrations (25mM) of MgCl2 inhibited adenylate cyclase activity determined in the presence of both corticotropin and GTP. At all MgCl2 concentrations, corticotropin and GTP activated the enzyme in a synergistic fashion. The magnitude of the stimulation of basal activity produced by corticotropin was a function of Mg2+ concentration, whereas that produced by GTP appeared largely independent of Mg2+ concentration. Adenylate cyclase activity in the bovine adrenal membrane was half-maximally stimulated by corticotropin concentrations in the range 0.3—1.0 nM. The concentration of corticotropin evoking half-maximum response was not significantly affected by raising the free Mg2+ concentration from 0.4 to 4.9 mM, nor by the presence of GTP. In the presence of GTP, high concentrations (over 1 micrometer) of corticotropin inhibited adenylate cyclase activity, although no inhibition was apparent in the absence of guanine nucleotide.

scholarly journals 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 β2-adrenoceptor-expressing NG108-15 cells

1995 ◽  
Vol 308 (1) ◽  
pp. 275-281 ◽  
Author(s):  
G D Kim ◽  
I C Carr ◽  
G Milligan
Keyword(s):  
Adenylate Cyclase ◽  
Nucleotide Binding ◽  
Dose Effect ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Guanine Nucleotide Binding ◽  
Dose Dependent ◽  
Stimulation Of

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.

Regulation of PTH receptor-adenylate cyclase system of canine kidney: influence of Mn2+ on effects of Ca2+, PTH, and GTP

1982 ◽  
Vol 242 (5) ◽  
pp. F457-F462
Author(s):  
E. Bellorin-Font ◽  
J. Tamayo ◽  
K. J. Martin
Keyword(s):  
Enzyme Activity ◽  
Adenylate Cyclase ◽  
Metal Ions ◽  
Guanine Nucleotides ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Adenylate Cyclase System ◽  
Guanine Nucleotide ◽  
Hormone Binding ◽  
Stimulation Of

Metal ions play important roles in the regulation of the activation of adenylate cyclase. Previous studies have suggested that an important site of action of metal ions is at or closely related to the nucleotide regulatory protein. The present studies examine the nature of the regulation of enzyme activity by divalent cations and the influence of Mn2+ on hormone binding and stimulation of adenylate cyclase. Studies were performed in canine renal cortical membranes. Substitution of Mg2+ by Mn2+ was associated with a progressive decline in the ability of GTP or PTH to stimulate adenylate cyclase activity. Mn2+ did not alter specific binding of an iodinated PTH analogue. However, in spite of the loss of guanine nucleotide stimulation of enzyme activity, the effects of guanine nucleotide on PTH binding were not altered in the presence of Mn2+. Substitution of Mg2+ by Mn2+ abolished the inhibitory effect of Ca2+ on basal adenylate cyclase activity. Similarly, the effects of GTP or PTH to enhance the inhibitory effects of Ca2+ on enzyme activity were abolished in the presence of Mn2+. Since Mg2+ and Ca2+ compete for a common allosteric site and Mn2+ abolished the effects of these cations, it would appear that Mn2+ also competes for the binding site of Mg2+ and Ca2+. The present studies demonstrating that Mn2+ does not affect hormone binding or the actions of guanine nucleotides on hormone binding yet totally eliminates the effect of GTP on enzyme activity indicate that the effect of Mn2+ occurs at the level of the interactions of the nucleotide regulatory component with the catalytic unit. In addition, these data suggest that there are two functionally distinct sites of guanine nucleotides with different ionic requirements.

scholarly journals Agonist regulation of adenylate cyclase activity in neuroblastoma × glioma hybrid NG108-15 cells transfected to co-express adenylate cyclase type II and the β2-adrenoceptor. Evidence that adenylate cyclase is the limiting component for receptor-mediated stimulation of adenylate cyclase activity

1996 ◽  
Vol 318 (3) ◽  
pp. 1033-1039 ◽  
Author(s):  
David J. MACEWAN ◽  
Gun-Do KIM ◽  
Graeme MILLIGAN
Keyword(s):  
Adenylate Cyclase ◽  
Partial Agonist ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Intrinsic Activity ◽  
Type Ii ◽  
Guanine Nucleotide ◽  
Maximal Output ◽  
Adenylate Cyclase Stimulation ◽  
Stimulation Of

Stable cell lines, derived from NG108-15 cells and transfected to express both the β2-adrenoceptor and adenylate cyclase type II, were produced and examined. The absence of adenylate cyclase type II in the parental cells and its presence in these clones was demonstrated by reverse transcriptase-PCR. Total cellular levels of adenylate cyclase were increased in a number of clones between 3- and 8-fold, as assessed by guanine nucleotide-stimulated specific high-affinity binding of [3H]forskolin to cellular membranes. Basal adenylate cyclase activity was markedly elevated compared with a clone expressing similar levels of the β2-adrenoceptor in the absence of adenylate cyclase type II. Each of NaF, forskolin and guanosine 5´-[β,γ-imido]triphosphate (a poorly hydrolysed analogue of GTP) produced substantially higher levels of adenylate cyclase activity in membranes of the clones positive for expression of adenylate cyclase type II than was achieved with the parental cells. Both isoprenaline, acting at the introduced β2-adrenoceptor, and iloprost, acting at the endogenously expressed IP prostanoid receptor, stimulated adenylate cyclase activity to much higher levels in the clones expressing adenylate cyclase type II compared with the clone lacking this adenylate cyclase; however, the concentration–effect curves for adenylate cyclase stimulation by these two agonists were not different between parental cells and clones overexpressing adenylate cyclase type II. A maximally effective concentration of the β-adrenoceptor partial agonist ephedrine displayed similar intrinsic activity and potency to stimulate adenylate cyclase in membranes of clones both with and without adenylate cyclase type II. Both secretin and 5´-N-ethylcarboxamidoadenosine (acting at an endogenous A2 adenosine receptor) were also able to produce substantially greater maximal activations of adenylate cyclase in the clones expressing excess adenylate cyclase type II, without alterations in agonist intrinsic activity or potency. These results demonstrate that the maximal output of the stimulatory arm of the adenylate cyclase cascade can be increased by increasing total levels of adenylate cyclase in the genetic background of NG108-15 cells.

Stimulation of Thyroid Adenylate Cyclase Activity in Sera of Patients with Nonthyroidal Illness

1986 ◽  
pp. 385-389
Author(s):  
F. Kakezono ◽  
S. Yamashita ◽  
N. Yokoyama ◽  
S. Morita ◽  
S. Okamoto ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Nonthyroidal Illness ◽  
Stimulation Of

scholarly journals Forskolin refractoriness. Exposure to the diterpene alters guanine nucleotide-dependent adenylate cyclase and calcium-uptake activity of cells cultured from the rat aorta

1987 ◽  
Vol 241 (2) ◽  
pp. 463-467 ◽  
Author(s):  
J F Krall ◽  
N Jamgotchian
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Cultured Cells ◽  
Rat Aorta ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Morphological Properties ◽  
Guanine Nucleotide ◽  
Intact Cells ◽  
Uptake Activity

Cells with the morphological properties of endothelial cells were cultured from the rat aorta. The cultured cells accumulated 45Ca2+ from the medium in a manner which was stimulated by forskolin and by 8-bromo-cyclic AMP. Pretreating the cultures for 20 h with forskolin diminished forskolin-dependent Ca2+-uptake activity. Adenylate cyclase activity of cultured cell homogenates was stimulated by guanosine 5′-[beta, gamma-imido]triphosphate (p[NH]ppG) and forskolin, and by isoprenaline in the presence, but not in the absence, of guanine nucleotide. p[NH]ppG increased forskolin sensitivity and caused a leftward shift in the forskolin dose-response curve. Pretreating the cultured cells with forskolin for 20 h, conditions that decreased forskolin-dependent Ca2+ uptake, increased basal and guanine nucleotide-dependent adenylate cyclase activity, but not forskolin-dependent activity determined in the absence of p[NH]ppG. Forskolin pretreatment diminished p[NH]ppG's capacity to increase forskolin sensitivity, but did not have a significant effect on either the sensitivity of adenylate cyclase to p[NH]ppG or its responsiveness to isoprenaline. These results suggest that the Ca2+-uptake mechanism is cyclic AMP-dependent and that guanine nucleotides mediated forskolin-dependent cyclic AMP production by the intact cells. In addition, there may be different guanine nucleotide requirements for hormone-receptor coupling and forskolin activation.

Prostaglandin E2-binding sites and cAMP production in porcine fundic mucosa

1981 ◽  
Vol 241 (4) ◽  
pp. G313-G320
Author(s):  
B. L. Tepperman ◽  
B. D. Soper
Keyword(s):  
Adenylate Cyclase ◽  
Prostaglandin E2 ◽  
Binding Site ◽  
Binding Sites ◽  
Biologically Active ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Scatchard Analysis ◽  
Fundic Mucosa ◽  
Stimulation Of

Biologically active [3H]prostaglandin E2 (PGE2) bound rapidly and specifically to membrane fractions from hog fundic mucosa. Optimal binding occurred in the 30,000-g membrane preparation at 37 degrees C (pH 5.0). Scatchard analysis of specific PgE2 binding revealed the presence of a heterogeneous population of binding sites with Kd values and binding site concentrations of approximately 1 X 10(-9) M and 1 fmol/mg prot and 2 X 10(-8) M and 20 fmol/mg prot, respectively. Specific binding was inhibited by the following agents in descending order of potency: PGE1, PGA2, PGD2, 6-keto-PGF1 alpha, and thromboxane B2. Trypsin treatment or boiling reduced or abolished specific PGE2 binding. PGE2 stimulated cAMP formation in the 2,500-g fraction, with an approximate Km of 1 X 10(-6) M, but stimulation of adenylate cyclase activity by PG was not evident in the 16,000-g or 30,000-g tissue preparations. These results suggest that a specific PGE2-binding site exists in the 16,000-g and 30,000-g fractions of porcine fundic mucosa, although an increase in cAMP-forming capacity could not b of 1 X 10(-6) M, but stimulation of adenylate cyclase activity by PG was not evident in the 16,000-g or 30,000-g tissue preparations. These results suggest that a specific PGE2-binding site exists in the 16,000-g and 30,000-g fractions of porcine fundic mucosa, although an increase in cAMP-forming capacity could not b of 1 X 10(-6) M, but stimulation of adenylate cyclase activity by PG was not evident in the 16,000-g or 30,000-g tissue preparations. These results suggest that a specific PGE2-binding site exists in the 16,000-g and 30,000-g fractions of porcine fundic mucosa, although an increase in cAMP-forming capacity could not be localized in these fractions in vitro.

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