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 Insulin activates the plasma-membrane and dense-vesicle cyclic AMP phosphodiesterase in hepatocytes by distinct routes

1983 ◽  
Vol 216 (1) ◽  
pp. 245-248 ◽  
Author(s):  
S R Wilson ◽  
A V Wallace ◽  
M D Houslay
Keyword(s):  
Plasma Membrane ◽  
Cyclic Amp ◽  
Insulin Receptor ◽  
Proteinase Inhibitors ◽  
Atp Depletion ◽  
Peripheral Plasma ◽  
Membrane Enzyme ◽  
Membrane Bound ◽  
Subsequent Activation ◽  
Distinct Membrane

Insulin elicits the activation of two distinct membrane-bound cyclic AMP phosphodiesterases when incubated at 37 degrees C for 5 min with intact hepatocytes: the ‘dense-vesicle’ enzyme and the peripheral-plasma-membrane enzyme. In hepatocytes the lysosomotropic agents chloroquine, methylamine and NH4Cl, as well as intracellular ATP depletion elicited by fructose or incubation with insulin at 22 degrees C, blocks selectively the activation of the ‘dense-vesicle’ enzyme. Incubation of hepatocytes with bacitracin, leupeptin and a variety of proteinase inhibitors failed to affect insulin's activation of these two cyclic AMP phosphodiesterases by distinct routes. It is suggested that activation of the ‘dense-vesicle’ enzyme occurs through a pathway triggered by the endocytosis, processing and recycling of the insulin receptor. This might involve the delivery, with subsequent activation, of a latent phosphodiesterase into this fraction.

scholarly journals Paradoxical stimulation by 1-methyl-3-isobutylxanthine of rat liver cyclic AMP phosphodiesterase activity

FEBS Letters ◽  
1982 ◽  
Vol 142 (2) ◽  
pp. 251-254 ◽  
Author(s):  
Christophe Erneux ◽  
Françoise Miot ◽  
Jean-Marie Boeynaems ◽  
Jacques E. Dumont
Keyword(s):  
Cyclic Amp ◽  
Rat Liver ◽  
Phosphodiesterase Activity ◽  
Cyclic Amp Phosphodiesterase
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