Insulin activates a rat liver peripheral plasma-membrane cyclic AMP phosphodiesterase by a phosphorylation mechanism

1980 ◽  
Vol 8 (5) ◽  
pp. 537-538 ◽  
Author(s):  
ROBERT J. MARCHMONT ◽  
MILES D. HOUSLAY
Keyword(s):  
Plasma Membrane ◽  
Cyclic Amp ◽  
Rat Liver ◽  
Cyclic Amp Phosphodiesterase ◽  
Peripheral Plasma ◽  
Phosphorylation Mechanism

scholarly journals Specific antibodies and the selective inhibitor ICI 118233 demonstrate that the hormonally stimulated ‘dense-vesicle’ and peripheral-plasma-membrane cyclic AMP phosphodiesterases display distinct tissue distributions in the rat

1987 ◽  
Vol 248 (3) ◽  
pp. 897-901 ◽  
Author(s):  
N J Pyne ◽  
N Anderson ◽  
B E Lavan ◽  
G Milligan ◽  
H G Nimmo ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Plasma Membrane ◽  
Cyclic Amp ◽  
Rat Liver ◽  
White Adipose Tissue ◽  
Tissue Homogenate ◽  
Phosphodiesterase Activity ◽  
Cyclic Amp Phosphodiesterase ◽  
Peripheral Plasma ◽  
Membrane Enzyme

Polyclonal-antibody preparations DV1 and PM1, raised against purified preparations of rat liver insulin-stimulated ‘dense-vesicle’ and peripheral-plasma-membrane cyclic AMP phosphodiesterases, were used to analyse rat liver homogenates by Western-blotting techniques. The antibody DV1 identified only the 63 kDa native subunit of the ‘dense-vesicle’ enzyme, and the antibody PM1 only the 52 kDa subunit of the plasma-membrane enzyme. These antibodies also detected the subunits of these two enzymes in homogenates of kidney, heart and white adipose tissue from rat. Quantitative immunoblotting demonstrated that the amount of these enzymes (by wt.) varied in these different tissues, as did the expression of these two enzymes, relative to each other, by a factor of as much as 7-fold. The ratio of the dense-vesicle enzyme to the peripheral-plasma-membrane enzyme was lowest in liver and kidney and highest in heart and white adipose tissue. ICI 118233 was shown to inhibit selectively the ‘dense-vesicle’ cyclic AMP phosphodiesterase in liver. It did this in a competitive fashion, with a Ki value of 3.5 microM. Inhibition of tissue-homogenate cyclic AMP phosphodiesterase activity by ICI 118233 was used as an index of the contribution to activity by the ‘dense-vesicle’ enzyme. By this method, a tissue distribution of the ‘dense-vesicle’ enzyme was obtained which was similar to that found by using the immunoblotting technique. The differential expression of isoenzymes of cyclic AMP phosphodiesterase activity in various tissues might reflect a functional adaptation, and may provide the basis for the different physiological actions of compounds which act as selective inhibitors.

scholarly journals Insulin stimulates the tyrosyl phosphorylation and activation of the 52 kDa peripheral plasma-membrane cyclic AMP phosphodiesterase in intact hepatocytes

1989 ◽  
Vol 261 (3) ◽  
pp. 897-904 ◽  
Author(s):  
N J Pyne ◽  
W Cushley ◽  
H G Nimmo ◽  
M D Houslay
Keyword(s):  
Plasma Membrane ◽  
Cyclic Amp ◽  
Insulin Treatment ◽  
Prior Treatment ◽  
High Affinity ◽  
Phosphorylation Of Proteins ◽  
Cyclic Amp Phosphodiesterase ◽  
Peripheral Plasma ◽  
Phosphoamino Acid Analysis ◽  
Tyrosyl Phosphorylation

The 52 kDa subunit of the peripheral-plasma-membrane insulin-stimulated high-affinity cyclic AMP phosphodiesterase can be specifically detected by the antibody PM1 by Western-blotting procedures and also can be immunoprecipitated from a hepatocyte extract. PM1-mediated immunoprecipitation from hepatocyte extracts showed that insulin treatment of intact 32P-labelled hepatocytes caused the rapid phosphorylation of the peripheral-plasma-membrane cyclic AMP phosphodiesterase. Phosphoamino acid analysis and the use of a phosphotyrosine-specific antibody indicated that phosphorylation occurred on tyrosyl residue(s) of this phosphodiesterase. Prior treatment of hepatocytes with glucagon (10 nM) completely blocked the insulin-mediated tyrosyl phosphorylation of this 52 kDa protein, as detected with both the PM1 and the anti-phosphotyrosine antibodies. Treatment of hepatocytes with glucagon alone did not increase the phosphorylation state of the peripheral-plasma-membrane cyclic AMP phosphodiesterase. The specific anti-phosphotyrosine antibody also detected the insulin-stimulated phosphorylation of proteins of 180 kDa, 95 kDa and 39 kDa. Prior treatment of hepatocytes with glucagon decreased the ability of insulin to phosphorylate the 180 kDa and 39 kDa species, but not the 95 kDa species.

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 Extracellular calcium modulates insulin's action on enzymes controlling cyclic AMP metabolism in intact hepatocytes

1993 ◽  
Vol 293 (1) ◽  
pp. 249-253 ◽  
Author(s):  
F Irvine ◽  
A V Wallace ◽  
S R Sarawak ◽  
M D Houslay
Keyword(s):  
Plasma Membrane ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Incubation Medium ◽  
Extracellular Calcium ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Cyclic Amp Phosphodiesterase ◽  
Peripheral Plasma ◽  
Incubation Buffer

Absence of physiological concentrations of extracellular Ca2+ in the Krebs-Henseleit incubation buffer did not affect the ability of 10 nM glucagon (< 5%) to increase hepatocyte intracellular cyclic AMP concentrations, but severely ablated (by approximately 70%) the ability of 10 nM insulin to decrease these elevated concentrations. Cyclic AMP metabolism is determined by production by adenylate cyclase and degradation by cyclic AMP phosphodiesterase (PDE). In the absence of added extracellular Ca2+ (2.5 mM), insulin's ability to activate PDE activity was selectively compromised, showing a failure of insulin to activate two of the three insulin-stimulated activities, namely the ‘dense-vesicle’ and peripheral plasma-membrane (PPM) PDEs. In the absence of added Ca2+, insulin's ability to inhibit adenylate cyclase activity in intact hepatocytes was decreased dramatically. Vasopressin and adrenaline (+ propranolol) failed to elicit the activation of either the ‘dense-vesicle’ or the PPM-PDEs. The presence of physiological concentrations of extracellular Ca2+ in the incubation medium is shown to be important for the appropriate generation of insulin's actions on cyclic AMP metabolism.

scholarly journals Properties and regulation of a trans-plasma membrane redox system in rat liver

1982 ◽  
Vol 204 (3) ◽  
pp. 795-801 ◽  
Author(s):  
Michael G. Clark ◽  
Eric J. Partick ◽  
Frederick L. Crane
Keyword(s):  
Plasma Membrane ◽  
Cyclic Amp ◽  
Rat Liver ◽  
Redox System ◽  
Isolated Perfused Rat Liver ◽  
Ferricyanide Reduction ◽  
Plasma Membrane Redox System ◽  
Glucose Release ◽  
Plasma Membrane Redox ◽  
Ph Values

1. Regulation of the reduction of ferricyanide by the isolated perfused rat liver was studied. 2. The rate of reduction was dependent on the rate of supply of ferricyanide and independent of perfusate oxygen concentration. 3. The effect of pH was also examined; the rate of reduction was optimal at pH 7.4 and was inhibited to a greater extent by pH values below 7.4 than those above 7.4. 4. The effects of substrates on the rate of ferricyanide reduction was assessed. Reductants of the cytosolic and mitochondrial NADH/NAD+ couple were tested. 2-Hydroxybutyrate (10mm), lactate (10mm), glycerol (10mm) and ethanol (10mm) each had no effect. Dihydroxyacetone (10mm) stimulated the rate. 5. Dehydroascorbate (1mm), stimulated the rate of ferricyanide reduction; the stimulation did not appear to be attributable to the production of reduced substances that were excreted to reduce extracellular ferricyanide. 6. The effects of glucagon and cyclic AMP on the rate of ferricyanide reduction were examined. Glucagon inhibited the rate by approx. 30% and half-maximal inhibition occurred at 0.1 nm, corresponding to the concentration at which half-maximal stimulation of glucose release occurred. Cyclic AMP stimulated glucose release but had no significant effect on the rate of ferricyanide reduction. It is concluded that the trans-plasma membrane redox system of liver that reduces extracellular ferricyanide is regulated by glucagon. The rate is also altered by the substrate dihydroxyacetone. The effect of glucagon may be direct as it cannot be mimicked by cyclic AMP and it occurs directly following exposure to the hormone.

scholarly journals N 6-(Phenylisopropyl)adenosine prevents glucagon both blocking insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase and uncoupling hormonal stimulation of adenylate cyclase activity in hepatocytes

1984 ◽  
Vol 222 (1) ◽  
pp. 177-182 ◽  
Author(s):  
A V Wallace ◽  
C M Heyworth ◽  
M D Houslay
Keyword(s):  
Plasma Membrane ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Phosphodiesterase Inhibitor ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity ◽  
Maximal Effect ◽  
Hormonal Stimulation ◽  
Cyclic Amp Phosphodiesterase ◽  
Phenylisopropyl Adenosine

Glucagon (10nM) prevented insulin (10nM) from activating the plasma-membrane cyclic AMP phosphodiesterase. This effect of glucagon was abolished by either PIA [N6-(phenylisopropyl)adenosine] (100nM) or adenosine (10 microM). Neither PIA nor adenosine exerted any effect on the plasma-membrane cyclic AMP phosphodiesterase activity either alone or in combination with glucagon. Furthermore, PIA and adenosine did not potentiate the action of insulin in activating this enzyme. 2-Deoxy-adenosine (10 microM) was ineffective in mimicking the action of adenosine. The effect of PIA in preventing the blockade by glucagon of insulin's action was inhibited by low concentrations of theophylline. Half-maximal effects of PIA were elicited at around 6nM-PIA. It is suggested that adenosine is exerting its effects on this system through an R-type receptor. This receptor does not appear to be directly coupled to adenylate cyclase, however, as PIA did not affect either the activity of adenylate cyclase or intracellular cyclic AMP concentrations. Insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase, in the presence of both glucagon and PIA, was augmented by increasing intracellular cyclic AMP concentrations with either dibutyryl cyclic AMP or the cyclic AMP phosphodiesterase inhibitor Ro-20-1724. PIA also inhibited the ability of glucagon to uncouple (desensitize) adenylate cyclase activity in intact hepatocytes. This occurred at a half-maximal concentration of around 3 microM-PIA. However, if insulin (10 nM) was also present in the incubation medium, PIA exerted its action at a much lower concentration, with a half-maximal effect occurring at around 4 nM.

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