Phosphorylation of rat C6 glioma cell DNA-dependent RNA polymerase II in vivo. Identification of phosphorylated subunits and modulation of phosphorylation by isoproterenol and N6,O2'-dibutyryl cyclic AMP

Evidence is presented that isoproterenol treatment of rat C6 glioma cells, under conditions that increase glioma cell cAMP levels, causes the phosphorylative modification of several RNA polymerase II subunits. RNA polymerase II in control and isoproterenol-stimulated 32Pi-labeled confluent glioma cells was immunoprecipitated from ribonuclease-treated nuclear extracts with hen anti-calf RNA polymerase II antiserum conjugated to Sepharose. The immunoprecipitated RNA polymerase II was analyzed for 32P-labeled subunits by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Using this technique, we have shown that isoproterenol causes a time-dependent increase of phosphate incorporation into RNA polymerase II subunits of 214,000, 180,000, 140,000, 35,000, 28,000, and 16,500 daltons. Phosphate incorporation occurred exclusively on serine in all of the six subunits. About 0.5-2 mol of phosphate/mol of RNA polymerase II subunit were incorporated. Dibutyryl cAMP (10(-3)M) mimics the stimulatory action of isoproterenol and mediates increased phosphate incorporation into the six subunits. (RS)-propranolol (10(-4)M) prevents the isoproterenol-mediated phosphorylative changes. These data indicate that isoproterenol, via cAMP, mediates a transient structural modification of RNA polymerase II subunits in rat C6 glioma cells which may possibly lead to a modulation of RNA polymerase II function(s).     

Stimulation of cyclic AMP in chondrocyte cultures: effects on sulfated-proteoglycan synthesis

The effects of N6,O2'-dibutyryladenosine 3':5'-cyclic monophosphate (DBcAMP), 8-bromoadenosine 3':5'-cyclic monophosphate (8Br-cAMP), 3':5'-cyclic monophosphate (cAMP), L-isoproterenol and L-epinephrine on sulfated-proteoglycan synthesis by rabbit articular chondrocytes were compared. DBcAMP and 8Br-cAMP in the presence or absence of 3-isobutyl-1-methylxanthine (IBMX) stimulated sulfated-proteoglycan biosynthesis after 20 h of incubation. cAMP had no significant effect. Both DBcAMP and 8Br-cAMP increased the hydrodynamic size of the newly synthesized proteoglycan monomer (A1D1) relative to control cultures. By contrast, although isoproterenol and epinephrine stimulated total cAMP synthesis, neither stimulated sulfated-proteoglycan synthesis. Whereas intracellular cAMP accumulated after incubation with DBcAMP and 8Br-cAMP, this was not the case with isoproterenol whether IBMX was present or not. Thus, stimulation of sulfated-proteoglycan synthesis by cAMP analogues in chondrocyte cultures appears to be dependent on increased intracellular cAMP accumulation rather than total cAMP biosynthesis.     

Regulation of Glycerol Phosphate Dehydrogenase and Lactate Dehydrogenase Activity by Forskolin and Dibutyryl Cyclic AMP in the C6 Glial Cells

We have compared the effects of norepinephrine, forskolin, and dibutyryl cyclic AMP (Bt2cAMP) on the regulation of the cytosolic enzyme glycerol phosphate dehydrogenase (GPDH) in the C6 rat glioma cell line. Forskolin and Bt2cAMP elicit a dose-dependent increase in the levels of the enzyme that was, however, unaffected by norepinephrine. The half-maximal effect of forskolin was obtained at 7-8 microM, and the effect was maximal at 30 microM. Dexamethasone at a 50 nM concentration produced a two- to sixfold induction of GPDH after 48 h. The combination of dexamethasone with forskolin or Bt2cAMP leads to an elevation in GPDH levels that is higher than that produced by one of the compounds alone. This potentiation is found when both agents are added together with or after the glucocorticoid. The increase in uninduced and dexamethasone-induced GPDH activity was blocked by cycloheximide and actinomycin D, indicating that de novo protein and RNA synthesis are required. The activity of cytosolic lactate dehydrogenase activity did not change after incubation with dexamethasone, but increased with forskolin or Bt2cAMP.     

Poly(adenosine diphosphate ribose) polymerase activity and nicotinamide adenine dinucleotide in differentiating cardiac muscle.

Poly(ADP-ribose) polymerase activity in nuclei isolated from differentiating cardiac muscle of the rat has been characterized and its activity measured during development. Optimum enzyme activity is observed at pH 8.5. Poly(ADP-ribose) polymerase is inhibited by ATP, thymidine, nicotinamide, theophylline, 3-isobutyl-1-methylxanthine and caffeine and stimulated by actinomycin D. The activity measured under optimal assay conditions increases during differentiation of cardiac muscle and is inversely related to the rate of DNA synthesis and to the activities of DNA polymerase alpha and thymidine kinase. When DNA synthesis and the activity of DNA polymerase alpha are inhibited in cardiac muscle of the 1-day-old neonatal rat by dibutyryl cyclic AMP or isoproterenol, the specific activity of poly(ADP-ribose) polymerase measured in isolated nuclei is increased. The concentration of NAD+ in cardiac muscle increases during postnatal development. In the adult compared with the 1-day-old neonatal rat the concentration of NAD+ relative to fresh tissue weight, DNA or protein increased 1.7-fold, 5.2-fold or 1.4-fold respectively. The concentration of NAD+ in cardiac muscle of the 1-day-old neonatal rat can be increased by approx. 20% by dibutyryl cyclic AMP. These data suggest that NAD+ and poly(ADP-ribose) polymerase may be involved with the repression of DNA synthesis and cell proliferation in differentiating cardiac muscle.     

Antioxidant defense against antidepressants in C6 and 1321N1 cells

The effects of pretreatment with the antioxidants reduced glutathione (GSH), ascorbate (ASC), Trolox (TROL), and combined ascorbate and Trolox (ASC/TROL) exposure on the acute (24 h) toxicities (EC50 value) of the antidepressants amitriptyline, imipramine (tricyclic antidepressants), fluoxetine (a selective serotonin reuptake inhibitor; SSRI), and tranylcypromine (a monoamine oxidase inhibitor; MAOI) were determined in the rat (C6) glioma and human (1321N1) astrocytoma cell lines using the neutral red uptake assay. The effects of pretreatment with buthionine-[S, R]-sulfoximine (BSO), and manipulation of intracellular cyclic AMP (cAMP) using isoproterenol (beta-receptor agonist), 3-isobutyl-1-methylxanthine (IBMX; a phosphodiesterase inhibitor), and dibutyryl cyclic AMP (dBcAMP; cAMP analogue) on antidepressant toxicity were also determined. Protective responses were observed after antioxidant treatments and manipulation of cAMP in both C6 cells pretreated with dBcAMP (+dBcAMP) and 1321N1 cells not pretreated with dBcAMP (-dBcAMP), with a few exceptions in 1321N1 cells (-dBcAMP). Some protective responses occurred in C6 cells (-dBcAMP) and 1321N1 cells (+dBcAMP) after isoproterenol and combined IBMX/isoproterenol pretreatment but not after just IBMX pretreatment. Pretreatment with BSO enhanced toxicity with the exception of fluoxetine. The antidepressants caused increases in intracellular GSH in the C6 cells at subcytotoxic concentrations, with decreases in GSH occurring at higher concentrations. Cytotoxicity of the antidepressants may be partly mediated through oxidative stress with alterations in signal transduction pathways.     

Calcium metabolism and amylase release in rat parotid acinar cells.

1. A method is described for the isolation of rat parotid acinar cells by controlled digestion of the gland with trypsin followed by collagenase. As judged by Trypan Blue exclusion, electron microscopy, water, electrolyte and ATP concentrations and release of amylase and lactate dehydrogenase, the cells are morphologically and functionally intact. 2. A method was developed for perifusion of acinar cells by embedding them in Sephadex G-10. Release of amylase was stimulated by adrenaline (0.1-10muM), isoproternol (1 or 10 MUM), phenylephrine (1 muM), carbamoylcholine (0.1 or 1 muM), dibutyryl cycle AMP (2 MM), 3-isobutyl-1-methylxanthine (1mM) and ionophore A23187. The effects of phenylephrine, carbamoylcholine and ionophore A23187 required extracellular Ca2+, whereas the effects of adrenaline and isoproterenol did not. 3. The incorporation of 45Ca into parotid cells showed a rapidly equilibrating pool (1-2 min) corresponding to 15% of total Ca2+ and a slowly equilibrating pool (greater than 3h) of probably a similar dimension. Cholinergic and alpha-adrenergic effectors and ionophore A23187 and 2,4-dinitrophenol increased the rate of incorporation of 45Ca into a slowly equilibrating pool, whereas beta-adrenergic effectors and dibutyryl cyclic AMP were inactive. 4. The efflux of 45Ca from cells into Ca2+-free medium was inhibited by phenylephrine and carbamoylcholine and accelerated by isoproterenol, adrenaline (beta-adrenergic effect), dibutyryl cyclic AMP and ionophore A23187. 5. A method was developed for the measurement of exchangeable 45Ca in mitochondria in parotid pieces. Incorporation of 45Ca into mitochondria was decreased by isoproterenol, dibutyryl cyclic AMP or 2,4-dinitrophenol, increased by adrenaline, and not changed significantly by phenylephrine or carbamoylcholine. Release of 45Ca from mitochondria in parotid pieced incubated in a Ca2+-free medium was increased by isoproterenol, adrenaline, dibutyryl cyclic AMP or 2,4-dinitrophenol and unaffected by phenylephrine or carbamoylcholine. 6. These findings are compatible with a role for Ca2+ as a mediator of amylase-secretory responses in rat parotid acinar cells, but no definite conclusions about its role can be drawn in the absence of knowledge of the molecular mechanisms involved, their location, and free Ca2+ concentration in appropriate cell compartment(s).     

Amylase secretion from saponin-permeabilized parotid cells evoked by cyclic AMP

Adenosine 3',5'-monophosphate (cAMP) evoked amylase release from saponin-permeabilized parotid cells of the rat. Saponin concentration was optimal at 10 micrograms/ml. Amylase release was stimulated by cAMP almost as well in Ca2+-free medium containing 1 mM EGTA as in the medium containing a physiological concentration of calcium. Although the basal and stimulated releases of amylase were markedly reduced by the further addition of 5 mM EGTA, the effect of cAMP was still detectable. The half-maximal dose of cAMP was 0.3 mM, whereas those of dibutyryl cAMP and 8-bromo-cAMP were 10-fold lower than that of cAMP. In the presence of 10 microM 3-isobutyl-1-methylxanthine, the half-maximal dose of cAMP was also decreased by 5-fold. These results suggest: 1) intracellular calcium is not essential for the exocytosis of amylase stimulated by cAMP; 2) the responsiveness of the cells to exogenous cAMP is reduced by phosphodiesterase.     

Regulation by Dibutyryl Cyclic AMP of Carnosine Synthesis in Astroglia-Rich Primary Cultures Kept in Serum-Free Medium

The synthesis of carnosine (beta-Ala-His) by astroglia-rich primary cultures was much higher if the cells were cultivated in Ham's nutrient mixture F-12 than if they were grown in Dulbecco's modified Eagle's medium. Carnosine synthesis was not affected by the presence of insulin, transferrin, phorbol myristate acetate, or dexamethasone. However, dibutyryl cyclic AMP and other agents that can, directly or indirectly, activate cyclic AMP-dependent protein kinases strongly lower the rate of carnosine synthesis. The depression of carnosine synthesis was dependent on the concentration of dibutyryl cyclic AMP. The effect was maximal (approximately 80% inhibition) in cultures preincubated with 1 mM dibutyryl cyclic AMP for 4 days. The adenylate cyclase activator forskolin, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, and 8-bromo-cyclic AMP caused the same depression as dibutyryl cyclic AMP, whereas neither butyrate nor dibutyryl cyclic GMP elicited any effect.     

Modulation of lipoprotein lipase activity in cultured rat mesenchymal heart cells and preadipocytes by dibutyryl cyclic AMP, cholera toxin and 3-isobutyl-1-methylxanthine

We have compared the effects of cellular cyclic AMP modulation on the regulation of lipoprotein lipase in cultures of rat epididymal pad preadipocytes and mesenchymal heart cells. Addition of dibutyryl cyclic AMP (dibutyryl cAMP) or 3-isobutyl-1-methylxanthine (IBMX) to preadipocytes grown in serum-containing culture medium resulted in a progressive decrease in lipoprotein lipase activity released into the culture medium so that at 6-8 h enzyme activity ranged between 20 and 30% of that recovered in the control dishes. Similar short-term (6-8 h) studies of the heart cell cultures showed a variable and much less pronounced depression of lipoprotein lipase activity. Thus, following dibutyryl cAMP and IBMX treatment, lipoprotein lipase activity ranged between 70 and 95% of control values. Incubation for 6 h with cholera toxin was followed by a 4-fold rise in the concentration of cellular cyclic AMP in both types of culture, but while in heart cell cultures enzyme activity was unchanged, lipoprotein lipase activity in preadipocytes decreased to 30% of control value. After 24 h incubation with all three effectors, an increase in lipoprotein lipase activity was seen. In the preadipocytes the increase ranged between 50 and 150% above control value, in the heart cell cultures it was 100-250%. 24-h incubation of heart cell cultures with dibutyryl cAMP resulted in a 6-fold increase of heparin-releasable lipoprotein lipase activity while residual activity was doubled. The rise in surface-bound lipoprotein lipase was evidenced also by an increase in the lipolysis of chylomicron triacylglycerol. In the presence of cycloheximide, the dibutyryl cAMP-induced heparin-releasable and residual lipoprotein lipase activity declined at the same rate as the basal activity. The reason for the difference in response of cultured preadipocytes and heart cells to the effectors during the first 8 h of incubation has not been elucidated, but could be related to a possible absence of hormone-sensitive lipase in the heart cells, and hence in a difference in intracellular metabolism of triacylglycerol. On the other hand, a common mechanism can be postulated for the long-term effect of cyclic AMP on the induction of lipoprotein lipase activity in both types of cultures. It probably involves mRNA and protein synthesis, which culminates in an increase in enzyme activity.     

Inhibitory effect of cyclic AMP on phorbol ester-stimulated production of reactive oxygen metabolites in rat glomeruli

Studies were conducted to investigate cross-talk between protein kinase C (PKC) and cyclic AMP (cAMP) pathways using rat glomeruli (Glm). Phorbol 12-myristate 13-acetate (PMA), a PKC activator, stimulated production of reactive oxygen metabolites (ROM) in Glm. Forskolin and dibutyryl cAMP (Bt2cAMP) inhibited production of ROM dose-dependently. In the presence of both Bt2cAMP and 3-isobutyl-1-methylxanthine (IBMX) an additive effect was observed. Forskolin at 10(-4) inhibited translocation of PKC from the cytosol to the membrane. These results demonstrate that cAMP-mediated inhibition can occur at a step distal to PKC activation.     

Effects of verapamil on lipolysis due to dibutyryl cyclic AMP.

The effects of verapamil, a calcium antagonist, on lipolysis in isolated rat adipocytes were studied. Verapamil (100 microM) potentiated lipolysis due to dibutyryl cyclic AMP (Bt2cAMP) at submaximal concentrations, with or without extracellular Ca2+. Lipolysis due to 0.5 mM-Bt2cAMP was potentiated by verapamil in a dose-dependent manner up to 200 microM, whereas at concentrations higher than 100 microM the stimulatory effect of verapamil was progressively diminished with or without extracellular Ca2+. Verapamil showed only an inhibitory effect on lipolysis due to adrenaline (0.1-10 microM) or 3-isobutyl-1-methylxanthine (IBMX; 25-200 microM). The stimulatory effect of verapamil on lipolysis due to Bt2cAMP was not blocked by alpha-adrenergic antagonists. These results suggest (i) that verapamil has a biphasic effect on lipolysis due to Bt2cAMP and only an inhibitory effect on that due to adrenaline or IBMX, and (ii) that extracellular Ca2+ or alpha-adrenergic receptors are not involved in the action of verapamil.     

Cyclic AMP effects on chloride transport and carbonic anhydrase activity in frog skin.

Unidirectional (36Cl) chloride fluxes across isolated and short-circuited frog skin were measured, with both sides bathed in low chloride solution. Transepithelial chloride influx was inhibited by exogenous cAMP as well as by substances enhancing its cellular concentration, such as epinephrine, isoproterenol, and 3-isobutyl-1-methylxanthine (IBMX). Epinephrine and isoproterenol addition resulted in an increase of transepithelial chloride outflux, but exogenous cAMP or IBMX had no significant effect on this unidirectional flux. Phenylephrine had no significant effect on influx or outflux. Carbonic anhydrase (CA) activity in extracts obtained from frog skin epithelium was inhibited by pretreatment with IBMX at 4-5 degrees C and prolonged exposure to cAMP at freezing point. cAMP or IBMX alone had no significant effects on CA activity. This catalytic activity was chloride insensitive and was abolished by 0.1 microM acetazolamide. Results suggest a Cl(-)-HCO3- exchange inhibition by cAMP via carbonic anhydrase inactivation. Chloride outflux stimulation by beta-adrenergic agonists does not seem to depend solely on an increase in cAMP concentration.     

Epidermal growth factor potentiates cyclic AMP accumulation in A-431 cells.

Epidermal growth factor (EGF) treatment of A-431 cells potentiates up to 5-fold the intracellular cyclic AMP (cAMP) accumulation induced by isoproterenol, cholera toxin, forskolin, or 3-isobutyl-1-methylxanthine (IBMX). EGF potentiates cAMP accumulation in several epithelial cell lines which overexpress the EGF receptor including A-431 cells, HSC-1 cells, and MDA-468 cells, and in the A-431-29S clone which expresses a normal complement of EGF receptors. Although EGF potentiates cAMP accumulation, EGF by itself does not measurably alter the basal level of cAMP. EGF rapidly enhances cAMP accumulation (within 1 to 3 min) in A-431 cells treated with these cAMP-elevating agents. EGF potentiation of cAMP accumulation does not reflect enhancement of beta-adrenergic receptor activation and is not a consequence of intracellular cAMP elevation or the concomitant activation of cAMP-dependent protein kinase. Since EGF potentiates accumulation of both intracellular and extracellular cAMP in isoproterenol-treated A-431 cells, EGF does not potentiate intracellular cAMP accumulation by inhibition of cAMP export. EGF potentiation of cAMP accumulation is pertussis toxin-insensitive and does not result from EGF inhibition of cAMP degradation in A-431 cells. These results demonstrate that EGF transmembrane signaling includes an interaction with a component of the adenylate cyclase system and that this interaction stimulates cAMP synthesis resulting in enhancement of cAMP accumulation.     

Isoproterenol produces a rapid increase in sialidase activity in rat heart tissue and cardiomyocyte-derived H9c2 cells in culture

The effects of isoproterenol on sialidase activity in rat cardiomyocytes were examined. Administration of isoproterenol to rats (0.2 or 2 mg/kg body weight) produced an increase in sialidase activity in total membrane fraction of heart tissue within 120 min (121+/-13% of the control at 120 min after administration of 0.2 mg isoproterenol/kg, n=5, P<0.05). Sialidase activity in cardiomyocyte-derived H9c2 cells was also increased by treatment with isoproterenol (10 microM) for 60 min. The effect of isoproterenol on sialidase activity was amplified by the addition of 3-isobutyl-1-methylxanthine (IBMX). Sialidase activity in H9c2 cells was elevated by treatment with dibutyryl cAMP plus IBMX without isoproterenol. The content of N-acetylneuraminic acid in cells decreased by 22% after treatment with isoproterenol plus IBMX. These results suggest that sialidase activity in rat cardiomyocytes is regulated by beta-adrenergic stimulators via a cAMP-dependent process. The increased activity of sialidase may account for the reduction of sialic acid content of cells.     

Cyclic adenosine 3',5'-monophosphate analogues modulate rat placental cell growth and differentiation

Cyclic adenosine 3',5'-monophosphate (cAMP) has been implicated in the control of placental function. The present investigation was designed to evaluate the actions of cAMP analogues on the control of rat placental development. Two model systems were used to assess the actions of cAMP in the placenta: 1) a rat placental cell line and 2) rat labyrinth placental explants. Elevation of intracellular cAMP via treatment with cAMP analogues, 3-isobutyl-1-methylxanthine, forskolin, or cholera toxin inhibited placental cell DNA synthesis whereas treatment with an analogue to cyclic guanosine 3',5'-monophosphate was without effect. The inhibitory actions of dibutyryl cAMP on DNA synthesis were at least partially reversible and were not the result of metabolic toxicity. Dibutyryl cAMP had dramatic effects on the organization and morphology of placental cells growing in vitro and diminished the ability of the placental cells to grow following transplantation into allogeneic hosts. Differentiation-associated characteristics of rat placental cells were also affected by cAMP. cAMP analogues stimulated placental cell progesterone release and inhibited placental cell alkaline phosphatase activity. Dibutyryl cAMP had effects on placental labyrinth explants similar to its effects on the placental cell line. Dibutyryl cAMP inhibited explant outgrowth while stimulating explant release of progesterone. In summary, cAMP effectively modulates the growth and differentiation of rat placental cells in vitro.     

The synergistic interaction of hydrocortisone and dibutyryl cyclic AMP during enzyme induction in hybrids between rat C6 glioma cells and FU5AH hepatoma cells

The hormone-responsive enzymes tyrosine aminotransferase and glycerol-3-phosphate dehydrogenase were studied with respect to current models of the mechanism of glucocorticoid/cAMP interaction during the induction of enzyme activity in responsive cell hybrids between rat C6 glioma cells and rat FU5AH hepatoma cells. The results of experiments involving protein and mRNA synthesis inhibitors, sequential addition of inducers, and the assay of cyclic-AMP-dependent protein kinase could not be adequately explained by any one model of inducer interaction. Comparison of the hybrid clones revealed the presence of factors that may modify induction but that are not essential for synergistic induction.     

Differential effects of Ro 20-1724 and isobutylmethylxanthine on the basal force of contraction and beta-adrenoceptor-mediated response in the rat ventricular myocardium.

Effects of Ro 20-1724, a selective inhibitor of soluble cGMP-insensitive type IV phosphodiesterase, on the force and cAMP levels were compared with those of 3-isobutyl-1-methylaxanthine, a non-selective inhibitor, in the rat ventricular myocardium. Ro 20-1724 scarcely affected the basal force of contraction and cAMP levels, whereas it enhanced the positive intropic effect and cAMP accumulation induced by isoproterenol more effectively than 3-isobutyl-1-methylxanthine. These results imply that inhibition of the soluble cGMP-insensitive type IV PDE by Ro 20-1724 may be crucially involved in the regulation of myocardial contractility through the interaction with cAMP generation in the rat ventricular myocardium.     

Biochemical consequences of follicle-stimulating hormone binding to testicular macrophages in culture

The present studies demonstrate that testicular macrophages respond to follicle-stimulating hormone (FSH) by: 1) stimulating the rate of incorporation of amino acids into secreted proteins; 2) increasing the rate of incorporation of uridine into RNA; and 3) enhancing the accumulation of intracellular cyclic adenosine monophosphate (cAMP; which was potentiated by the addition of 1 mM 3-isobutyl-1-methylxanthine; MIX). In addition, dibutyryl cAMP (dbcAMP) enhanced the incorporation of amino acids into secreted proteins; however, this cAMP analog had no effect on the incorporation of uridine into RNA. Finally, it was demonstrated that testicular macrophages possess specific receptors with a high affinity for FSH.     

Beta-adrenergic stimulation enhances translocation, processing and synthesis of lipoprotein lipase in rat heart cells

Cells isolated from newborn rat hearts were cultured for 10-14 days, and lipoprotein lipase activity was present in an intracellular and heparin-releasable pool. Treatment of the cultures with 10(-7) M isoproterenol for 3 min resulted in a 3-fold increase in heparin-releasable lipoprotein lipase and a concomitant decrease in residual cellular enzyme activity. Similar results were obtained by treatment with dibutyryl cAMP. Treatment with isoproterenol or dibutyryl cAMP for 2 h affected glycosylation of immunoadsorbable lipoprotein lipase, so that the ratio of [3H]galactose to [14C]mannose in the heparin-releasable enzyme increased from 3.8 (control) to 13.0 (isoproterenol-treated). The change in the ratio of the sugars in the cellular fraction of the enzyme was from 3.1 to 9.9. 2 h treatment with isoproterenol did not enhance new enzyme synthesis, as determined by incorporation of [3H]leucine into immunoadsorbable lipoprotein lipase. 24 h after addition of either isoproterenol or dibutyryl cAMP to the culture medium, stimulation of enzyme synthesis was demonstrated. The present results permit three effects of isoproterenol on lipoprotein lipase to be distinguished: stimulation of translocation from a cellular to heparin-releasable pool; enhanced processing of mannose residues and terminal glycosylation; stimulation of synthesis of enzyme protein.