Investigation of the subcellular distribution of cyclic-AMP phosphodiesterase in rat hepatocytes, using a rapid immunological procedure for the isolation of plasma membrane

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.

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Effects of Dexamethasone and Glucagon after Long-Term Exposure on Cyclic AMP Phosphodiesterase 4 in Cultured Rat Hepatocytes

Cellular Signalling ◽

10.1016/s0898-6568(99)00039-x ◽

1999 ◽

Vol 11 (9) ◽

pp. 685-690 ◽

Cited By ~ 14

Author(s):

Thomas Hermsdorf ◽

Wito Richter ◽

Dietrich Dettmer

Keyword(s):

Cyclic Amp ◽

Rat Hepatocytes ◽

Phosphodiesterase 4 ◽

Cyclic Amp Phosphodiesterase ◽

Cultured Rat Hepatocytes

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Cross-talk between glucagon- and adenosine-mediated signalling systems in rat hepatocytes: effects on cyclic AMP-phosphodiesterase activity

Biochemical Journal ◽

10.1042/bj3120763 ◽

1995 ◽

Vol 312 (3) ◽

pp. 763-767 ◽

Cited By ~ 14

Author(s):

M Robles-Flores ◽

G Allende ◽

E Piña ◽

J A García-Sáinz

Keyword(s):

Cyclic Amp ◽

Pertussis Toxin ◽

Plasma Membranes ◽

Rat Hepatocytes ◽

Dependent Manner ◽

Phosphodiesterase Activity ◽

Cyclic Amp Phosphodiesterase ◽

Cyclic Amp Accumulation ◽

A1 Adenosine Receptors ◽

Dose Dependent

The effect of adenosine analogues on glucagon-stimulated cyclic AMP accumulation in rat hepatocytes was explored. N6-Cyclopentyladenosine (CPA), 5′-N-ethylcarboxamidoadenosine and N6-(R-phenylisopropyl)adenosine inhibited in a dose-dependent manner the cyclic AMP accumulation induced by glucagon. This effect seems to be mediated through A1 adenosine receptors. Pertussis toxin completely abolished the effect of CPA on glucagon-stimulated cyclic AMP accumulation in whole cells which suggested that a pertussis-toxin-sensitive G-protein was involved. On the other hand, this action of adenosine analogues on glucagon-induced cyclic AMP accumulation was reverted by the selective low-Km cyclic AMP-phosphodiesterase inhibitor Ro 20-1724. Analysis of cyclic AMP-phosphodiesterase activity in purified hepatocyte plasma membranes showed that glucagon in the presence of GTP inhibited basal PDE activity by 45% and that CPA reverted this inhibition in dose-dependent manner. In membranes derived from pertussis-toxin-treated rats, we observed no inhibition of cyclic AMP-phosphodiesterase activity by glucagon in the absence or presence of CPA. Our results indicate that in hepatocyte plasma membranes, stimulation of adenylate cyclase activity and inhibition of a low-Km cyclic AMP phosphodiesterase activity are co-ordinately regulated by glucagon, and that A1 adenosine receptors can inhibit glucagon-stimulated cyclic AMP accumulation by blocking glucagon's effect on phosphodiesterase activity.

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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

Biochemical Journal ◽

10.1042/bj2220177 ◽

1984 ◽

Vol 222 (1) ◽

pp. 177-182 ◽

Cited By ~ 12

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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