The phorbol ester TPA inhibits cyclic AMP phosphodiesterase activity in intact hepatocytes

Treatment of intact hepatocytes with the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) potentiated the ability of glucagon to increase intracellular cyclic AMP concentrations. This effect was dose-dependent upon TPA, exhibiting an EC50 of 0.39 ng/ml and such activation was observed at both saturating and sub-saturating concentrations of glucagon. However, this stimulatory effect of TPA was completely abolished by the presence of the cyclic AMP phosphodiesterase inhibitor 1-isobutyl-3-methylxanthine, when TPA now inhibited the glucagon-stimulated increase in intracellular cyclic AMP concentrations. It is suggested that, as well as inhibiting glucagon-stimulated adenylate cyclase activity, TPA also inhibits cyclic AMP phosphodiesterase activity in intact hepatocytes. Treatment of either hepatocyte homogenates or purified cyclic AMP phosphodiesterase with TPA failed to show any direct inhibitory effect of TPA on activity showing that TPA did not exert any direct inhibitory action on phosphodiesterase activity. However, homogenates made from hepatocytes that had been pre-treated with TPA did show a reduced cyclic AMP phosphodiesterase activity. It is suggested that TPA might inhibit cyclic AMP phosphodiesterase activity through phosphorylation by C-kinase.     

N6-(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.

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.     

Evidence that the peripheral cyclic AMP phosphodiesterase of rat liver plasma membranes is a metalloenzyme.

Cyclic nucleotide phosphodiesterase activity in salt extracts of rat liver plasma membranes was progressively inactivated by treatment with the metal chelators 8-hydroxyquinoline and o-phenanthroline, but not the non-chelating m-phenanthroline isomer. Activity at 20 microM-cyclic AMP was lost more slowly than activity at 0.4 microM-cyclic AMP. The activity of treated preparations was partially restored by incubation with Zn2+ or Mn2+ ions (in the presence of 1 mM-MgCl2) but not with Ca2+, Cd2+, Co2+, Cu2+ or Fe2+ ions, nor by MgCl2 alone. The results suggest the presence in the membrane extracts of a cyclic AMP phosphodiesterase containing tightly bound metal, possibly Zn or Mn, that affects the enzyme's affinity for cyclic AMP.     

Mobilization of intracellular calcium by glucagon and cyclic AMP analogues in isolated rat hepatocytes

Separate or combined addition of cyclic AMP-dependent and Ca2+-linked hormones to isolated rat hepatocytes suspended in a low Ca2+ medium reduced the total cellular Ca. When the hormones were administered together, their effects were not additive. This suggests that both types of hormones mobilize Ca2+ from a common intracellular pool. In the presence of 1.8 mM extracellular Ca2+, the Ca2+ influx counterbalanced or even exceeded the hormone-induced Ca2+ loss, depending on the ability of the hormones to stimulate the Ca2+ influx.     

A peripheral and an intrinsic enzyme constitute the cyclic AMP phosphodiesterase activity of rat liver plasma membranes.

1. Approx. 10% of the rat liver cellular cyclic AMP phosphodiesterase activity was associated with a plasma-membrane fraction. 2. Lineweaver-Burk plots of this activity were clearly non-linear, yielding extrapolated Km values of 0.7 and 60.6 microns. 3. Treatment of these membranes with high-ionic-strength NaCl solutions apparently released 80% of this activity assayed at 0.4 micron-cyclic AMP, and 15% of the activity assayed at 1 mM-cyclic AMP. 4. The high-salt-solubilized enzyme gave a non-linear Lineweaver-Burk plot. 5. The cyclic AMP phosphodiesterase activity of the washed high-salt-treated membranes exhibited a linear Lineweaver-Burk plot, yielding a Km of 60 microns. 6. The high-salt-solubilized enzyme exhibited a single peak of activity upon polyacrylamide-gel electrophoresis, a single peak upon sucrose-density-gradient centrifugation (3.9 S) and decayed as a single exponential upon heat-treatment (half-life 1 min at 55 degrees C). 7. The activity of washed high-salt-treated membranes decayed as a single exponential upon heat-treatment (half-life 42 min at 55 degrees C), and was solubilized in the detergent Triton X-100. 8. Cytosol-derived cyclic AMP phosphodiesterase activity could bind to washed high-salt-treated plasma membranes, but was totally eluted by washing with 1 mM-KHCO3, unlike the high-salt-solubilized enzyme, which required high salt concentrations to elute it. 9. We suggest that the cyclic AMP phosphodiesterase activity of rat liver plasma membranes can be resolved into two components: a single peripheral protein exhibiting apparent negative co-operativity, that is distinct from cytosol forms, and an intrinsic protein exhibiting normal Michaelis kinetics.     

Cyclic AMP and cyclic GMP as the respective mediators of the intracycle stimulation of DNA synthesis and mitosis induced by glucagon and insulin in primary neonatal rat hepatocytes

Within a wide range of concentrations ($\text{i.e.}$, from 10^?15 to 10^?8 mole/1), equimolar mixtures of dibutyryl-cyclic AMP and dibutyryl-cyclic GMP or of glucagon and dibutyryl-cyclic GMP or of insulin and dibutyryl-cyclic AMP faithfully mimicked the stimulation of DNA-synthetic and mitotic activities elicited by equimolar associations of glucagon and insulin in 4-to-5-day-old neonatal rat hepatocytes in primary tissue culture. These observations strongly suggest that the intracycle, growth-promoting effects of the two pancreatic hormones are mediated via both purine cyclic nucleotides in the neonatal rat hepatocytes.     

Inhibition of receptor-mediated stimulation of cyclic AMP accumulation in neuroblastoma-hybrid NCB-20 cells by a phorbol ester

Activation of protein kinase C by phorbol esters such as phorbol 12-myristate 13-acetate (PMA), modulates responsiveness of the cyclase system in many cell types. In the neuroblastoma-hybrid cell line NCB-20, PMA causes a reduction in receptor-mediated accumulation of cyclic AMP. The reduction in receptor responses by PMA occurs within 3 min and is still apparent at 40 min. This occurs in a concentration-dependent manner with an EC50 for PMA of approx. 30 nM. Accumulations of cyclic AMP that are elicited by prostaglandin E2, vasoactive intestinal peptide or 2-chloroadenosine are decreased in the presence of PMA. Accumulations of cyclic AMP that are elicited by forskolin in the absence of a receptor agonist are unaffected by the presence of PMA. Inhibition of cyclic AMP generation by dopamine is not diminished by PMA suggesting the receptor input through the inhibitory Ni-guanyl nucleotide binding protein is still functional after PMA treatment. The generalized inhibition of receptor-mediated responses by PMA could be due to a protein kinase C-mediated phosphorylation of the stimulatory Ns-guanyl nucleotide binding protein, but other mechanisms are possible.     

Effect of somatomedin A and insulin on cyclic AMP generation in isolated rat hepatocytes

Insulin and somatomedin A were shown to have inhibitory action on glucagon stimulated but not basal cyclic AMP production in isolated rat hepatocytes. The inhibition was dose-dependent and the potency per mol was about 100 fold higher for insulin than for somatomedin A.     

Acute in vivo stimulation of low-Km cyclic AMP phosphodiesterase activity by insulin in rat-liver Golgi fractions

A low-Km phosphodiesterase activity, which is acutely stimulated by insulin in vivo, has been identified in plasma membranes and Golgi fractions prepared from rat liver homogenates in isotonic sucrose. Within seconds after insulin injection (25 micrograms/100 g body weight) cAMP phosphodiesterase activity increases by 30-60% in Golgi fractions and by 25% in plasma membranes; activity in crude particulate and microsomal fractions is unaffected. The increase in activity is short-lived in the light and intermediate Golgi fractions, but persists for at least 10 min in the heavy Golgi fraction. It precedes the translocation of insulin and insulin receptors to these fractions, which is maximal at 5 min. The doses of insulin required for half-maximal and maximal activation are, respectively, 7.5 micrograms/100 g and 25 micrograms/100 g body weight. Golgi-associated cAMP phosphodiesterase activity shows non-linear kinetics; a high-affinity component (Vmax, 13 pmol min-1 mg protein-1; Km, 0.35 microM) is detectable. Insulin treatment increases the Vmax 60-70%, but does not affect the Km. Unlike the low-Km cAMP phosphodiesterase associated with crude particulate fractions, the Golgi-associated activity is not easily extractable by solutions of low or high ionic strength. On analytical sucrose density gradients, low-Km cAMP phosphodiesterase associated with the total particulate fraction equilibrates at lower densities than endoplasmic reticulum and lysosomal markers, but at a higher densities than plasma membrane, Golgi markers and insulin receptors. Insulin treatment increases the specific activity of the enzyme by 20-60% at densities below 1.12 g cm-3, and by 20-40% in the density interval 1.23-1.25 g cm-3. Such treatment also causes a slight, but significant shift in the distribution of phosphodiesterase towards lower densities. It is suggested that Golgi elements or physically similar subcellular structures are a major site of localization of insulin-sensitive cAMP phosphodiesterase in rat liver. However, internalization of the insulin-receptor complex is probably not required for enzyme activation.     

Purification and properties of the insulin-stimulated cyclic AMP phosphodiesterase from rat liver plasma membranes.

The peripheral high-affinity cyclic AMP phosphodiesterase from rat liver plasma membranes was purified to apparent homogeneity. The procedure used involved the initial purification of liver plasma membranes and the solubilization of the enzyme by using a high-ionic-strength medium. This was followed by chromatography of the enzyme on DEAE-cellulose, Affi-Gel Blue, a novel affinity column and Sephadex G-100. A 9500-fold purification of the enzyme with a 24% yield was achieved by this procedure. The purified enzyme was apparently monomeric (Mr 52000) as it exhibited identical molecular weights on analysis by gel filtration, sedimentation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It is suggested that the non-Michaelis kinetics exhibited by the enzyme are due to it obeying a mnemonical mechanism, where it displays Km 0.7 micrometer, Vmax. 9.1 units/mg of protein and Hill coefficient (h) 0.62. Cyclic GMP acts as a poor substrate for the enzyme, with Km 120 micrometer and Vmax. 0.4 unit/mg of protein, and also as an inhibitor of the enzyme, with I50 (concentration giving 50% inhibition) 150 micrometer when assayed at 0.4 micrometer-cyclic AMP. Inhibition by 5'-AMP is unlikely to be of physiological importance, as it is only a weak inhibitor of the enzyme (I50 47 mM assayed at 0.4 micrometer-cyclic AMP).     

Identification and characterization of both the cytosolic and particulate forms of cyclic GMP-stimulated cyclic AMP phosphodiesterase from rat liver.

Two enzymes displaying cyclic GMP-stimulated cyclic AMP phosphodiesterase activity were purified from rat liver to apparent homogeneity: a 'particulate enzyme' found as an integral membrane protein associated with the plasma membrane, and a 'soluble' enzyme found in the cytosol. The physical properties of these enzymes were very similar, being dimers of Mr 134,000, composed in each instance of two subunits of Mr = 66,000-67,000. Both enzymes showed similar kinetics for cyclic AMP hydrolysis. They are both high-affinity enzymes, with kinetic constants for the particulate enzyme of Km = 34 microM and Vmax. = 4.0 units/mg of protein and for the cytosolic enzyme Km = 40 microM and Vmax. = 4.8 units/mg of protein. In both instances hydrolysis of cyclic AMP appeared to show apparent positive co-operativity, with Hill coefficients (happ.) of 1.5 and 1.6 for the particulate and cytosolic enzymes respectively. However, in the presence of 2 microM-cyclic GMP, the hydrolysis of cyclic AMP obeyed Michaelis kinetics (happ. = 1) for both enzymes. The addition of micromolar concentrations of cyclic GMP had little effect on the Vmax. for cyclic AMP hydrolysis, but lowered the Km for cyclic AMP hydrolysis to around 20 microM in both cases. However, at low cyclic AMP substrate concentrations, cyclic GMP was a more potent activator of the particulate enzyme than was the soluble enzyme. The activity of these enzymes could be selectively inhibited by cis-16-palmitoleic acid and by arachidonic acid. In each instance, however, the hydrolysis of cyclic AMP became markedly more sensitive to such inhibition when low concentrations of cyclic GMP were present. Tryptic peptide maps of iodinated preparations of these two purified enzyme species showed that there was considerable homology between these two enzyme forms.     

Bidirectional concentration-dependent effects of glucagon and dibutyryl cyclic AMP on DNA synthesis in cultured adult rat hepatocytes

Glucagon and dibutyryl cyclic AMP exerted both stimulatory and inhibitory effects on hepatocyte DNA synthesis when added to primary monolayer cultures in the presence of serum, dexamethasone, insulin and epidermal growth factor. The stimulation occurred at low concentrations of glucagon (1 pM-1 nM) or dibutyryl cyclic AMP (1 nM-1 microM), while the agents inhibited DNA synthesis at higher concentrations (usually glucagon at over 10 nM or dibutyryl cyclic AMP at over 10 microM). The stimulatory effect was stronger at low cell densities (less than 20 X 10(3) hepatocytes/cm2). When the hepatocytes were cultured at higher densities, stimulatory effects were reduced or absent and the inhibition of (hormone-induced) DNA synthesis by a high concentration of glucagon was much more pronounced than at low cell densities. These results indicate dual, bidirectional, effects of cyclic AMP on hepatocyte DNA synthesis.     

An effect of glucagon on 3', 5'-cyclic AMP phosphodiesterase activity in isolated rat hepatocytes.

Glucagon was found to activate the low Km form of 3′,5′-cyclic AMP phosphodiesterase in intact isolated rat hepatocytes while the high Km phosphodiesterase was unaltered. Activation was concentration dependent and occurred at the same concentration required to observe an increase in 3′,5′-cyclic AMP levels in the cell. The maximal increase in activity occurred within 5 minutes of incubation with glucagon and was sustained for the 35 minutes assayed.     

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

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.     

Dynamics of calmodulin and cyclic AMP phosphodiesterase in plasma membranes of rat livers and ascites hepatomas

1. Plasma membranes from ascites hepatoma cells (AH-7974, AH-130) contained much smaller amounts of calmodulin (about half) and cyclic AMP phosphodiesterase (about one-third) compared to plasma membranes of rat livers. 2. Some of calmodulin molecules in liver plasma membranes were released by repeated washing. The 'washed' liver plasma membranes showed the presence of specific binding sites for externally added calmodulin molecules (bovine brain) (N = 140 pmol/mg protein, Kd = 7.9 . 10(-8) M). The calmodulin content of AH-7974 plasma membranes was not reduced by repeated washing. The binding of calmodulin to the 'washed' AH-7974 plasma membranes was only of nonspecific nature with negative cooperativity. 3. Plasma membranes (liver and AH-7974) appeared to contain both calmodulin-dependent and calmodulin-independent phosphodiesterase, but the stimulation by externally added Ca2+ plus calmodulin was rather small. Externally added calmodulin-dependent phosphodiesterase (bovine brain) was bound more to 'washed' liver plasma membranes than to 'washed' AH-7974 plasma membranes. Newly bound phosphodiesterase appeared to be more sensitive to the stimulation by Ca2+ plus calmodulin in 'washed' hepatoma plasma membranes than in 'washed' liver plasma membranes. 4. Preincubation of 'washed' plasma membranes (liver and hepatoma) with calmodulin did not affect the binding of phosphodiesterase, but the sensitivity of phosphodiesterase to the stimulation by Ca2+ plus calmodulin in hepatoma plasma membranes was lost.     

Role of microtubules in insulin and glucagon stimulation of amino acid transport in isolated rat hepatocytes

The effects of the microtubule inhibitor, colchicine, on insulin or glucagon stimulation of alpha-amino[1-14C]-isobutyric acid (AIB) transport were investigated in isolated hepatocytes from normal fed rats. Under all conditions tested, AIB uptake appeared to occur through two components of transport: a low affinity (Km approximately 50 mM) component and a high affinity (Km approximately 1 mM) component. Within 2 h of incubation, insulin and glucagon, at maximal concentrations, increase AIB (0.1 mM) uptake by 2- to 3-fold and 4- to 6-fold, respectively. Colchicine, at the low concentration of 5 X 10(-7) M, slightly reduces basal AIB transport, decreases by 80% the simulatory effect of insulin, and diminishes by 40% the stimulatory effect of either glucagon or dibutyryl cAMP. Kinetic analysis of AIB influx indicates that the drug inhibits the increase in Vmax of a high affinity (Km approximately 1 mM) component of transport stimulated by insulin or glucagon, without affecting the kinetic parameters of a low affinity component of transport (Km approximately 50 mM). Various short term hormonal effects of insulin and glucagon (changes in glucose, urea, and lactate production) were found not to be modified by the drug. Vinblastine elicits similar changes as colchicine on AIB uptake. Lumicolchicine, a colchicine analogue that does not bind to tubulin, has no effect. The concentration of colchicine (10(-7) M) required for half-maximal inhibition of hormone-stimulated AIB transport is in the appropriate range for specific microtubule disruption. These data suggest that microtubules are involved in the regulation of the insulin or glucagon stimulation of AIB transport in isolated rat hepatocytes.