scholarly journals Islet-activating protein blocks glucagon desensitization in intact hepatocytes

1984 ◽  
Vol 222 (1) ◽  
pp. 189-194 ◽  
Author(s):  
C M Heyworth ◽  
E Hanski ◽  
M D Houslay
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Inhibitory Effect ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Inhibitory Input ◽  
Pronounced Increase ◽  
Guanine Nucleotide Regulatory Protein

Treatment of intact hepatocytes with islet-activating protein, from Bordatella pertussis, led to a pronounced increase in the ability of glucagon to raise intracellular cyclic AMP concentrations. Islet-activating protein, however, caused no apparent increase in the intracellular concentration of cyclic AMP under basal conditions. These effects were attributed to an enhanced ability of adenylate cyclase, in membranes from hepatocytes treated with islet-activating protein, to be stimulated by glucagon. When forskolin was used to amplify the basal adenylate cyclase activity, elevated GTP concentrations were shown to inhibit adenylate cyclase activity in membranes from control hepatocytes. This inhibitory effect of GTP was abolished if the hepatocytes had been pre-treated with islet activating protein. In isolated liver plasma membranes, islet-activating protein caused the NAD-dependent ribosylation of a Mr-40000 protein, the putative inhibitory guanine nucleotide regulatory protein, Ni. This effect was inhibited if guanosine 5′-[beta‐thio]diphosphate rather than GTP was present in the ribosylation incubations. The ability of glucagon to uncouple or desensitize the activity of adenylate cyclase in intact hepatocytes was also blocked by pre-treating hepatocytes with islet-activating protein. Islet-activating protein thus heightens the response of hepatocytes to the stimulatory hormone glucagon. It achieves this by both inhibiting the expression of desensitization and also removing a residual inhibitory input expressed in the presence of glucagon.

scholarly journals Treatment of streptozotocin-diabetic rats with metformin restores the ability of insulin to inhibit adenylate cyclase activity and demonstrates that insulin does not exert this action through the inhibitory guanine nucleotide regulatory protein Gi

1988 ◽  
Vol 249 (2) ◽  
pp. 537-542 ◽  
Author(s):  
D Gawler ◽  
G Milligan ◽  
M D Houslay
Keyword(s):  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Diabetic Rats ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Guanine Nucleotide ◽  
Liver Plasma Membranes ◽  
Streptozotocin Diabetic Rats ◽  
Guanine Nucleotide Regulatory Protein

Insulin caused the inhibition of glucagon-stimulated adenylate cyclase activity in liver plasma membranes, but failed to inhibit this activity in liver membranes from rats made diabetic by treatment with either alloxan or streptozotocin. Treatment of streptozotocin-diabetic rats with insulin, to normalize their blood glucose concentrations, restored this action of insulin. Rats treated with the biguanide drug metformin exhibited a decreased content of the inhibitory guanine nucleotide regulatory protein Gi in liver plasma membranes assessed both structurally, by using a specific polyclonal antibody (AS7), and functionally. Treatment of normal rats with metformin did not alter insulin's ability to inhibit adenylate cyclase in liver plasma membranes; however, metformin treatment of streptozotocin-diabetic rats completely restored this inhibitory action of insulin. Liver plasma membranes from streptozotocin-diabetic animals which either had or had not been treated with metformin had contents of Gi which were less than 10% of those seen in control animals. We conclude that: (i) insulin does not inhibit adenylate cyclase activity through the inhibitory guanine nucleotide regulatory protein Gi; (ii) streptozotocin- and alloxan-induced diabetes elicit a selective insulin-resistant state; and (iii) metformin can exert a post-receptor effect, at the level of the liver plasma membrane, which restores the ability of insulin to inhibit adenylate cyclase.

scholarly journals Challenge of hepatocytes by glucagon triggers a rapid modulation of adenylate cyclase activity in isolated membranes

1983 ◽  
Vol 214 (1) ◽  
pp. 93-98 ◽  
Author(s):  
C M Heyworth ◽  
M D Houslay
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Regulatory Protein ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Maximum Effect ◽  
Guanine Nucleotide ◽  
Rapid Modulation ◽  
Guanine Nucleotide Regulatory Protein ◽  
Dose Dependent

Membrane fractions obtained from hepatocytes treated with glucagon exhibited a decreased glucagon (with or without GTP)-stimulated adenylate cyclase activity. A maximum effect was seen in around 5 min. No change in the rate of cyclic AMP production was observed for the basal, NaF-, p[NH]ppG (guanosine 5′-[beta, gamma-imido]-triphosphate)- and GTP-stimulated states of the enzyme. The lag observed in the p[NH]ppG-stimulated adenylate cyclase activity of native membranes was abolished when membranes from glucagon-pretreated cells were used. When Mn2+ replaced Mg2+ in the assays, the magnitude of the apparent desensitization was decreased. Mn2+ abolished the lag of onset of p[NH]ppG-stimulated activity in native membranes. The desensitization process was dose-dependent on glucagon, which exhibited a Ka of 4 X 10(-10) M. Depletion of intracellular ATP did not affect this process. It is suggested that this desensitization occurs at the level of the guanine nucleotide-regulatory protein.

scholarly journals The action of islet activating protein (pertussis toxin) on insulin's ability to inhibit adenylate cyclase and activate cyclic AMP phosphodiesterases in hepatocytes

1986 ◽  
Vol 235 (1) ◽  
pp. 145-149 ◽  
Author(s):  
C M Heyworth ◽  
A M Grey ◽  
S R Wilson ◽  
E Hanski ◽  
M D Houslay
Keyword(s):  
Plasma Membrane ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Pertussis Toxin ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Guanine Nucleotide ◽  
Cyclic Amp Phosphodiesterase ◽  
Peripheral Plasma ◽  
Guanine Nucleotide Regulatory Protein

Treatment of hepatocytes with islet activating protein (pertussis toxin) from Bordetella pertussis blocked the ability of insulin to inhibit adenylate cyclase activity both in broken plasma membranes and in intact hepatocytes. Such treatment of intact hepatocytes with pertussis toxin did not prevent insulin from activating the peripheral plasma membrane cyclic AMP phosphodiesterase although it did inhibit the ability of insulin to activate the ‘dense-vesicle’ cyclic AMP phosphodiesterase. The ability of glucagon pretreatment of hepatocytes to block insulin's activation of the plasma membrane cyclic AMP phosphodiesterase was abolished in pertussis toxin-treated hepatocytes. It is suggested that the ability of insulin to manipulate cyclic AMP concentrations by inhibiting adenylate cyclase and activating the plasma membrane and ‘dense-vesicle’ cyclic AMP phosphodiesterases involves interactions with the guanine nucleotide regulatory protein system occurring in liver plasma membranes.

scholarly journals Resensitization of lutropin-desensitized tumour Leydig-cell adenylate cyclase with human erythrocyte membranes

1982 ◽  
Vol 204 (2) ◽  
pp. 613-616 ◽  
Author(s):  
C J Dix ◽  
B A Cooke
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Human Erythrocyte ◽  
Plasma Membranes ◽  
Control Cell ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Erythrocyte Membranes ◽  
Cell Plasma ◽  
Human Erythrocyte Membranes

Purified rat tumour Leydig cells were pretreated with or without lutropin (1 h at 32 degrees C). The plasma membranes were then isolated and the adenylate cyclase activity measured in the presence of freshly prepared or heat-inactivated (1 h at 60 degrees C) human erythrocyte membranes. In plasma membranes from control cells in the presence of heat-inactivated human erythrocyte membranes both guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) plus lutropin and NaF caused a 45-50-fold increase in cyclic AMP production over 30 min compared with 12-13 fold p[NH[ppG and 2-3-fold with lutropin alone. In plasma membranes isolated from lutropin-pretreated cells the NaF- and the p[NH]ppG-stimulated cyclic AMP production rates were unchanged, but no effect of lutropin could be demonstrated with or without added p[NH]ppG. However, after mixing lutropin-desensitized Leydig tumour-cell plasma membranes with freshly prepared human erythrocyte plasma membranes, the adenylate cyclase activity in the presence of lutropin, p[NH]ppG, lutropin plus p[NH]ppG and NaF were similar to those of control cell plasma membranes treated in the same manner. The possible mechanisms of this reversal of lutropin-induced desensitization by human erythrocytes are discussed.

scholarly journals Insulin and glucagon attenuate the ability of cholera toxin to activate adenylate cyclase in intact hepatocytes

1988 ◽  
Vol 251 (2) ◽  
pp. 447-452 ◽  
Author(s):  
F J Irvine ◽  
M D Houslay
Keyword(s):  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Regulatory Protein ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Stimulatory Action ◽  
Maximal Activation ◽  
Lag Period ◽  
Guanine Nucleotide Regulatory Protein ◽  
Dose Dependent

Treatment of intact hepatocytes with cholera toxin at 37 degrees C caused a stable activation of adenylate cyclase activity after a lag period of around 10 min. The presence of either insulin (10 nM) or glucagon (10 nM) in the incubation medium had little effect on this lag period; however, these hormones markedly attenuated the maximal activation of adenylate cyclase activity that could be achieved by treatment with cholera toxin. Such actions of insulin and glucagon were dose-dependent, with EC50 values (concn. giving 50% inhibition) of 0.20 nM for insulin and 0.49 nM for glucagon, and were not additive. Treatment of intact hepatocytes with either glucagon or insulin did not affect the ability of cholera toxin to cause the ADP-ribosylation of the 45 kDa alpha-subunit of the stimulatory guanine nucleotide regulatory protein, Gs, in intact hepatocytes. It is suggested that treatment of intact hepatocytes with either insulin or glucagon attenuates the stimulatory action of ADP-ribosylated Gs on adenylate cyclase.

Characterization of a beta-adrenergic receptor in porcine trachealis muscle

1984 ◽  
Vol 247 (5) ◽  
pp. C342-C349 ◽  
Author(s):  
K. J. Popovich ◽  
C. Hiller ◽  
A. Hough ◽  
J. S. Norris ◽  
L. E. Cornett
Keyword(s):  
Smooth Muscle ◽  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Beta Agonist ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Beta Adrenergic ◽  
Adrenergic Antagonist ◽  
Trachealis Muscle

To establish a model of airway smooth muscle function we studied binding of [3H]dihydroalprenolol [( 3H]DHA), a beta-adrenergic antagonist, to membrane preparations of porcine trachealis muscle and investigated the response of adenylate cyclase to l-isoproterenol in tissue and plasma membranes. [3H]DHA binding was of high affinity (Kd = 1.0 +/- 0.1 nM), was saturable (Bmax = 87.6 +/- 13.2 fmol/mg protein), and was 90% beta 2 and 10% beta 1. Adenylate cyclase activity in the membrane preparation was (in pmol.10 min-1.mg protein-1 +/- SE): basal 420 +/- 74, guanosine 5'-triphosphate (GTP) (10 micron) 600 +/- 45, GTP (10 microM) + l-isoproterenol (100 microM) 660 +/- 63, NaF (10 mM) 1,500 +/- 134, and forskolin (100 microM) 3,000 +/- 410. Guanosine 5'-diphosphate (GDP) and GTP were active cofactors; l-isoproterenol appeared to function as an effector exchanging GTP for GDP on the guanine nucleotide regulatory protein. There was close agreement of the effective dose (ED50) of the l-isoproterenol-induced relaxation (0.95 +/- 0.45 microM) and the inhibitory constant of l-isoproterenol binding (0.39 +/- 0.10 microM). l-Isoproterenol (100 microM) induced a 100% increase in adenosine 3',5'-cyclic monophosphate (cAMP) levels in tissue strips over basal activity. Investigation of the difference in adenylate cyclase activity between tissue and plasma membranes revealed that l-isoproterenol responsive adenylate cyclase was diminished after initial homogenization. Electron microscopy demonstrated disruption of all cells at this early stage of preparation. The decrease in l-isoproterenol responsive adenylate cyclase following cell rupture is different from other tissues and suggests a difference in the actions of beta-agonist in smooth muscle compared with other tissues.

scholarly journals Structural specificity for prostaglandin effects on hepatocyte glycogenolysis

1990 ◽  
Vol 267 (1) ◽  
pp. 59-62 ◽  
Author(s):  
E P Brass ◽  
M J Garrity
Keyword(s):  
Glucose Metabolism ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Plasma Membranes ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Sprague Dawley ◽  
Hepatic Glucose Metabolism ◽  
Sprague Dawley Rats ◽  
Cyclic Amp Accumulation

Prostaglandins (PGs) are known to have effects on hepatic glucose metabolism. Some actions of PGs in intact liver systems may not involve PG effects directly at the level of the hepatocyte. To define the ability of structurally distinct prostaglandins to affect hepatocyte metabolism directly, the regulation of glycogenolysis was studied in hepatocytes isolated from male Sprague-Dawley rats. PGF and PGB2 inhibited glucagon-stimulated glycogenolysis in the hepatocyte system. Pinane thromboxane A2 (PTA2) and PGD2 had no effect on glucagon-stimulated glycogenolysis. Consistent with their inhibition of glucagon-stimulated glycogenolysis, PGF2 and PGF2 alpha inhibited glucagon-stimulated hepatocyte cyclic AMP accumulation. These actions of PGB2 and PGF2 alpha are identical with those previously reported for PGE2. Additionally, PGE2, PGF2 alpha and PGB2 inhibited glucagon-stimulated adenylate cyclase activity in purified hepatic plasma membranes. In contrast, PGF2 alpha, PGD2 and PTA2 were all without affect on basal rates of hepatocyte glycogenolysis or hepatocyte cyclic AMP content. PGE2 also inhibited glycogenolysis stimulated by the alpha-adrenergic agonist phenylephrine. Exogenous arachidonic acid was not able to reproduce the affects of PGE2 or PGF2 alpha on hepatocyte glycogenolysis, consistent with an extra-hepatocyte source of the prostaglandins in the intact liver. Thus PGE2 and PGF2 alpha act specifically to inhibit glucagon-stimulated adenylate cyclase activity. No prostaglandin tested was found to stimulate glycogenolysis. PGE2 and PGF2 alpha may represent intra-hepatic modulators of hepatocyte glucose metabolism.

Inhibitory effect of clonidine upon adenylate cyclase activity, cyclic AMP production, and insulin release in rat pancreatic islets

1984 ◽  
Vol 4 (6) ◽  
pp. 511-521 ◽  
Author(s):  
P. Garcia-Morales ◽  
S. P. Dufrane ◽  
A. Sener ◽  
I. Valverde ◽  
W. J. Malaisse
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Pancreatic Islets ◽  
Insulin Release ◽  
Inhibitory Action ◽  
Fold Increase ◽  
Inhibitory Effect ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Rat Pancreatic Islets

Conflicting opinions were recently expressed concerning the possible effect of α2-adrenergic agonists upon cyclic AMP production in pancreatic islets. In the present: study, clonidine inhibited glucose-induced insulin release from rat pancreatic islets, this inhibitory effect being abolished by idazoxan. Clonidine did not suppress the capacity of forskolin to augment glucose-induced insulin release. In a particulate subcellular fraction derived from the islets, adenylate cyclase was activated by calmodulin (in the presence of Ca2+), NaF, GTP, L-arginine, and forskolin, and slightly inhibited by clonidine. The inhibitory action of clonidine upon basal adenylate cyclase activity was more pronounced in islet crude homogenates. The inhibitory effect of clonidine was antagonized by forskolin whether in the particulate fraction or crude homogenate. At variance with the modest effects of glucagon, D-glucose, L-arginine, or a tumor-promoting phorbol ester upon cyclic AMP production by intact islets, forskolin caused a six-fold increase in cyclic AMP production. Clonidine inhibited cyclic AMP production by intact islets, whether in the absence or presence of forskolin. It is proposed that the inhibitory action of clonidine upon insulin release is attributable, in part at least, to inhibition of adenylate cyclase.

Esters of adenosine 5'-phosphate with lipoid hydroxy compounds (adenosine nucleolipids) and their effects on the activity of enzymes of cyclic AMP system

1979 ◽  
Vol 44 (5) ◽  
pp. 1645-1650
Author(s):  
Sixtus Hynie ◽  
Jiří Smrt
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Inhibitory Effect ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Amp System ◽  
Strong Inhibitory Effect ◽  
Activity Of Enzymes ◽  
Hydroxy Compounds

Esters of adenosine 5'-phosphate with lipoid hydroxy compounds exhibit strong inhibitory effect on adenylate cyclase activity. The activities of cyclic AMP phosphodiesterase and protein kinase are moderately inhibited.

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