Ontogeny of hepatic insulin and glucagon receptors and adenylate cyclase in rabbit

1983 ◽  
Vol 244 (6) ◽  
pp. E624-E631
Author(s):  
S. Ganguli ◽  
M. K. Sinha ◽  
B. Sterman ◽  
P. Harris ◽  
M. A. Sperling
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Regulatory Protein ◽  
Late Gestation ◽  
Camp Production ◽  
Hormonal Activation ◽  
Glucagon Receptors ◽  
Significant Increment

In rabbit liver plasma membranes (LPM), specific binding of 125I-insulin rapidly increased in late gestation and peaked at birth, declining thereafter. In contrast, 125I-glucagon binding was lowest in late gestation, somewhat higher at birth, and increased by 48 h although only to 20-25% of adult. These changes in binding were due to changing numbers of receptors involving predominantly high affinity sites for insulin and low affinity sites for glucagon, with only minor changes in affinity. Despite measurable glucagon receptors by birth, fetal LPM produced no increment above basal in cAMP production with maximal doses of glucagon (10(-6) M), prostaglandin E1 (10(-4) M), or epinephrine (10(-4) M). Near birth only NaF (10 mM) produced a modest but significant increment in cAMP. By 2 h postbirth, all stimuli evoked significant increments in cAMP production that increased progressively but was still only 15-20% of adult at 48 h. Furthermore, although specific binding of cholera toxin was greater in fetal LPM (11 +/- 1 vs. 6 +/- 1%), cholera toxin-stimulated cAMP production increased by only 12-26% above basal in the fetus compared with 220% in adult. Markers of membrane purity including 5'-nucleotidase, phosphodiesterase, and insulin or glucagon degradation were not different in fetus and adult. We conclude that receptors and components of the adenylate cyclase complex mature independently; initial coupling occurs between the G/F regulatory protein and the catalytic unit (NaF but not hormonal activation) followed within hours of birth by coupling to the hormone receptor.

scholarly journals Guanosine 5′-triphosphate and guanosine 5′-[βγ-imido]triphosphate effect a collision coupling mechanism between the glucagon receptor and catalytic unit of adenylate cyclase

1980 ◽  
Vol 186 (3) ◽  
pp. 649-658 ◽  
Author(s):  
Miles D. Houslay ◽  
Irene Dipple ◽  
Keith R. F. Elliott
Keyword(s):  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Regulatory Protein ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Lipid Phase ◽  
Trypsin Treatment ◽  
Phase Separations ◽  
Catalytic Unit

1. GTP, but not p[NH]ppG (guanosine 5′-[βγ-imido]triphosphate), abolishes the sensitivity of glucagon-stimulated adenylate cyclase to the lipid-phase separations occurring in the outer half of the bilayer in liver plasma membranes from rat. 2. When either GTP or p[NH]ppG alone stimulate adenylate cyclase, the enzyme senses only those lipid-phase separations occurring in the inner half of the bilayer. 3. Trypsin treatment of intact hepatocytes has no effect on the basal, fluoride-, GTP- or p[NH]ppG-stimulated adenylate cyclase activity. However, 125I-labelled-glucagon specific binding decays with a half-life matching that of the decay of glucagon-stimulated adenylate cyclase activity. 4. When GTP or p[NH]ppG are added to assays of glucagon-stimulated activity, the half-life of the trypsin-mediated decay of activity is substantially increased and the decay plots are no longer first-order. 5. Trypsin treatment of purified rat liver plasma membranes abolishes basal and all ligand-stimulated adenylate cyclase activity, and 125I-labelled-glucagon specific binding. 6. Benzyl alcohol activates the GTP- and p[NH]ppG-stimulated activities in an identical fashion, whereas these activities are affected differently when glucagon is present in the assays. 7. We suggest that guanine nucleotides alter the mode of coupling between the receptor and catalytic unit. In the presence of glucagon and GTP, a complex of receptor, catalytic unit and nucleotide regulatory protein occurs as a transient intermediate, releasing a free unstable active catalytic unit. In the presence of p[NH]ppG and glucagon, the transient complex yields a relatively stable complex of the catalytic unit associated with a p[NH]ppG-bound nucleotide-regulatory protein.

scholarly journals Evidence for the existence of an Ns-type regulatory protein in Trypanosoma cruzi membranes

1986 ◽  
Vol 237 (3) ◽  
pp. 913-917 ◽  
Author(s):  
C D Eisenschlos ◽  
A A Paladini ◽  
L Molina y Vedia ◽  
H N Torres ◽  
M M Flawiá
Keyword(s):  
Membrane Proteins ◽  
Trypanosoma Cruzi ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Regulatory Protein ◽  
Adenylate Cyclase System ◽  
Gtp Binding ◽  
Polypeptide Band

The existence of a GTP-binding protein of the Ns type in Trypanosoma cruzi was explored. Epimastigote membranes were labelled by cholera toxin in the presence of [adenine-14C]NAD+. After SDS/polyacrylamide-gel electrophoresis of extracted membrane proteins, a single labelled polypeptide band of apparent Mr approx. 45,000 was detected. Epimastigote cells were treated with N-ethylmaleimide and electrofused to lymphoma S49 cells lacking the Ns protein. Evidence indicates that in such electrofusion-generated cell hybrids a heterologous adenylate cyclase system was reconstituted with the Ns protein provided by T. cruzi epimastigotes.

Cholera-toxin-dependent ADP-ribosylation of the adenylate cyclase regulatory protein in turkey erythrocyte membranes

1981 ◽  
Vol 209 (1) ◽  
pp. 284-290 ◽  
Author(s):  
Robert W. Downs ◽  
Sharon A. Reen ◽  
Michael A. Levine ◽  
Gerald D. Aurbach ◽  
Allen M. Spiegel
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Regulatory Protein ◽  
Erythrocyte Membranes ◽  
Adp Ribosylation

scholarly journals Adenylate Cyclase Activation by Cholera Toxin in Pig Epidermis: An Obligatory Role of the GTP-Regulatory Protein

1983 ◽  
Vol 81 (2) ◽  
pp. 131-136 ◽  
Author(s):  
Junji Takeda ◽  
Kenji Adachi ◽  
Kenneth M. Halprin ◽  
Victor Levine ◽  
Clyde Woodyard
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Regulatory Protein ◽  
Cyclase Activation ◽  
Adenylate Cyclase Activation

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.

LH release is facilitated by agents that alter cyclic AMP-generating system

1984 ◽  
Vol 246 (1) ◽  
pp. E44-E51 ◽  
Author(s):  
M. J. Cronin ◽  
W. S. Evans ◽  
E. L. Hewlett ◽  
M. O. Thorner
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Regulatory Protein ◽  
Dependent Manner ◽  
Calcium Dependent ◽  
Camp Analogue ◽  
Lh Release ◽  
Integral Proteins ◽  
Generating System

The issue of whether the adenosine 3',5'-monophosphate (cAMP)-generating system contributes to luteinizing hormone (LH) release was addressed by using several complementary probes in vitro. Pertussis toxin is considered to modify covalently an inhibitory adenylate cyclase regulatory protein. Treatment of gonadotrophs with this toxin increased both basal LH release and the efficacy of gonadotropin-releasing hormone (GnRH)-stimulated LH release with no apparent effect on GnRH potency. Cholera toxin, which probably activates adenylate cyclase by covalently altering another regulatory protein, forskolin, which directly stimulates the catalytic subunit of adenylate cyclase, and the cAMP analogue 8-Br-cAMP amplified both basal LH release (in a dose-dependent manner) and GnRH-stimulated LH release after a lag of 1 (cholera toxin and 8-Br-cAmP) and 4 (forskolin) h. It is noteworthy that these belated effects occurred in spite of the fact that cellular cAMP accumulation was markedly increased within 30 min after cholera toxin and at 1 min after forskolin addition. There was no change in total radioimmunoassayable LH (cellular + released) in either the basal or GnRH-treated cells after cholera toxin and forskolin for up to 24 h. Finally, the forskolin-amplified LH release was reversible and calcium dependent because D-600, EDTA, and calcium-free medium inhibited this effect. These results, generated with three complementary probes that affect integral proteins of the adenylate cyclase complex, suggest a function for cAMP in modulating LH release.

Gluco- and mineralocorticoids control adenylate cyclase in specific nephron segments

1990 ◽  
Vol 258 (4) ◽  
pp. F812-F820 ◽  
Author(s):  
A. Doucet ◽  
C. Barlet-Bas ◽  
S. Siaume-Perez ◽  
C. Khadouri ◽  
S. Marsy
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Mechanism Of Action ◽  
Thick Ascending Limb ◽  
Aluminum Fluoride ◽  
Camp Production ◽  
Nephron Segments ◽  
Glucocorticoid Administration ◽  
Collecting Tubule ◽  
Adrenalectomized Rats

Adrenal insufficiency is associated with an impairment of kidney diluting and concentrating ability, defects that may result from alterations of vasopressin-induced adenosine 3',5'-cyclic monophosphate (cAMP) production. The purpose of this study were 1) to localize the sites of decreased vasopressin-stimulated adenylate cyclase (AC) activity along the nephron of adrenalectomized rats; 2) to determine whether the response of AC to other hormones is altered by adrenalectomy; 3) to evaluate whether changes in AC are due to the deficiency in mineralocorticoids and/or glucocorticoids; and 4) to characterize the mechanism of action of corticosteroids on the AC system. Results indicate that adrenalectomy reduced AC stimulation by vasopressin, glucagon, and calcitonin in the thick ascending limb, whereas only the response to vasopressin decreased in the collecting tubule. Glucocorticoid administration curtailed adrenalectomy-induced alterations of AC in the thick ascending limb, whereas that in the collecting tubule was prevented by mineralocorticoids. Adrenalectomy did not alter forskolin-stimulated AC, whereas it decreased responses to aluminum fluoride and cholera toxin. Finally, alterations of fluoride- and cholera toxin-stimulated AC were prevented by glucocorticoid and mineralocorticoid repletion in the thick ascending limb and collecting tubule, respectively.

scholarly journals Insulin inhibits the cholera-toxin-catalysed ribosylation of a Mr-25000 protein in rat liver plasma membranes

1985 ◽  
Vol 228 (3) ◽  
pp. 593-603 ◽  
Author(s):  
C M Heyworth ◽  
A D Whetton ◽  
S Wong ◽  
B R Martin ◽  
M D Houslay
Keyword(s):  
Cholera Toxin ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Target Cells ◽  
Guanine Nucleotide ◽  
Whole Cells ◽  
Kinase C ◽  
Fractionation Procedure ◽  
Rat Liver Plasma Membranes ◽  
Guanine Nucleotide Regulatory Protein

A method is described for preparing a plasma-membrane fraction from hepatocytes by a rapid, gentle, Percoll fractionation procedure. Cholera toxin elicited the ribosylation of a number of proteins in these membranes, including the components of the stimulatory guanine nucleotide regulatory protein, Ns. Insulin, however, inhibited the ability of cholera toxin to ribosylate a protein of Mr 25 000. The action was decreased in membranes from cells that had been pre-treated with glucagon. Ribosylation of both the components of Ns and the Mr-25 000 species occurred in whole cells treated with cholera toxin, because membranes from such treated cells exhibited decreased labelling when incubated with [32P]NAD+ and activated cholera toxin. The labelling of proteins, including the Mr-25 000 species, with [32P]NAD+ and cholera toxin in the plasma membranes was decreased by an inhibitor of ribosylation. Azido-GTP photoaffinity labelling identified several high-affinity GTP-binding proteins, including one of Mr 25 000. Cholera toxin failed to ribosylate the Mr-25 000 protein in membranes from cells that had been pre-treated with the tumour-promoting agent 12-O-tetradecanoylphorbol 13-acetate (TPA). In membranes from such treated cells, insulin actually allowed cholera toxin to label this species. As TPA activates protein kinase C, it is possible that the Mr-25 000 protein, or a species that interacts with it, is a substrate for phosphorylation. These observations may offer an explanation for some of the perturbing effects that TPA exerts on insulin's action. It is suggested that the insulin receptor interacts with the guanine nucleotide regulatory protein system in the liver, and that the Mr-25 000 species may be a component of Nin, a specific guanine nucleotide regulatory protein that has been proposed to mediate certain of the actions of insulin on target cells [Houslay & Heyworth (1983) Trends Biochem. Sci. 8, 449-452].

scholarly journals Binding of parathyroid hormone to bovine kidney-cortex plasma membranes

1973 ◽  
Vol 134 (4) ◽  
pp. 913-921 ◽  
Author(s):  
H. S. Sutcliffe ◽  
T. J. Martin ◽  
J. A. Eisman ◽  
R. Pilczyk
Keyword(s):  
Parathyroid Hormone ◽  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Specific Binding ◽  
Rat Kidney ◽  
Kidney Cortex ◽  
Bovine Kidney ◽  
Hormone Sensitive ◽  
Chloramine T

1. Plasma membranes were purified from bovine kidney cortex, with a fourfold increase in specific activity of parathyroid hormone-sensitive adenylate cyclase over that in the crude homogenate. The membranes were characterized by enzyme studies. 2. Parathyroid hormone was labelled with 125I by an enzymic method and the labelled hormone shown to bind to the plasma membranes and to be specifically displaced by unlabelled hormone. Parathyroid hormone labelled by the chloramine-t procedure showed no specific binding. 75Se-labelled human parathyroid hormone, prepared in cell culture, also bound to the membranes. 3. Parathyroid hormone was shown to retain biological activity after iodination by the enzymic method, but no detectable activity remained after chloramine-t treatment. 4. High concentration of pig insulin inhibited binding of labelled parathyroid hormone to plasma membranes and partially inhibited the hormone-sensitive adenylate cyclase activity in a crude kidney-cortex preparation. 5. EDTA enhanced and Ca2+ inhibited binding of labelled parathyroid hormone to plasma membranes. 6. Whereas rat kidney homogenates were capable of degrading labelled parathyroid hormone to trichloroacetic acid-soluble fragments, neither crude homogenates nor purified membranes from bovine kidney showed this property. 7. Binding of parathyroid hormone is discussed in relation to metabolism and initial events in hormone action.

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