Evidence for differential synthesis of thyroxine and tri-iodothyronine by cultured human thyroid cells following exposure to thyrotrophin or dibutyryl cyclic AMP.

Cultured human thyroid cells treated with thyrotrophin (TSH) or dibutyryl cyclic AMP release more tri-iodothyronine (T3) and thyroxine (T4) than unsupplemented cells. Column chromatography was used to investigate the secretion of newly-synthesised 125-I labelled T3 and T4 from cells cultured with 125-I and TSH or dibutyryl cyclic AMP. Radioimmunoassays were used to determine total T3 and T4 release from cells cultured with unlabelled iodide.Iodothyronines released after TSH addition contained more 125-I than those released after dibutyryl cyclic AMP. This increase in 125-I was primarily in “new” T4. Release of “new” T3, however, was increased more by dibutyryl cyclic AMP than by TSH. Dibutyryl cyclic AMP and TSH were comparable in their stimulation of total T3 and total T4 release.Interpretation of these observations suggests that TSH and dibutyryl cyclic AMP may differ in some aspects of their in vitro effects on cellular iodination and iodothyronine coupling systems.     

The effect of cyclic AMP on glycoprotein secretion in isolated rat hepatocytes

The effects of dibutyryl cyclic AMP on glycoprotein biosynthesis, intracellular mobilization, and secretion in isolated rat hepatocytes are described. Dibutyryl cyclic AMP (2.5 mm) initially suppresses [³H]glucosamine or [³H]fucose incorporation into cellular macromolecular material; however, after $\text{3}\text{1}\text{2}$ h, the incorporation of these radiolabeled carbohydrates into macromolecular material was stimulated relative to control cells. The stimulation in accumulation of cellular glycoprotein occurred in membrane-associated fractions, with most of this accumulation occurring in the Golgi elements. The glycoprotein produced in the presence of dibutyryl cyclic AMP was quantitatively precipitated by antibodies directed against rat serum, suggesting that the accumulated cellular material is normally destined for secretion from the cell. Dibutyryl cyclic AMP also produced a drastic inhibition of glycoprotein secretion which persisted during the cellular accumulation of glycosylated material. Exposure of the hepatocytes to colchicine (10 μm) produced a similar increase in accumulation of [³H]glucosamine-containing immunoprecipitable material in the cellular fraction and a similar inhibition in secretion. The initial dibutyryl cyclic AMP-mediated suppression of synthesis of intracellular glycosylated material occurred entirely in non-membrane-associated intracellular fractions. Also, the initial accumulation of [³H]glucosamine-containing immunoprecipitable material was not suppressed during the first $\text{3}\text{1}\text{2}$ h after exposure to dibutyryl cyclic AMP, suggesting the initial suppression represents a metabolic process unrelated to secretion. The incorporation of [³H]leucine into macromolecular material was inhibited in both cellular and secreted fractions after exposure to dibutyryl cyclic AMP; however, the accumulation into the extracellular environment was inhibited to a greater extent. The patterns of [³H]glucosamine-containing lipid biosynthesis were unaffected by dibutyryl cyclic AMP.     

Cyclic AMP and growth of Ehrlich ascites tumor cells. Lack of cyclic AMP elevation in nutritionally deprived cells and mechanism of retardation of growth by dibutryl cyclic AMP.

Cyclic AMP levels in Ehrlich ascites tumor cells changed little after deprivation of cells of essential nutrients, serum, glucose and amino acids, deprival of each of which leads to marked inhibition of growth and protein synthesis. Cyclic AMP levels also changed little after the addition of these nutrients to deprived cells. Thus cyclic AMP is not likely to be the intracellular mediator for growth regulation by these three nutrients. Elevation of cyclic AMP levels for short periods by exposure of cells to choleratoxin or theophylline produced only slight changes in parameters of protein synthesis (polyribosome pattern and rate of [3H]leucine incorporation). An exposure for 1 day to dibutyryl cyclic AMP did not inhibit cell growth. However, prolonged exposure to dibutyryl cyclic AMP inhibited the multiplication of Ehrlich ascites cells both in suspension and in stationary cultures. No morphological effects were evident in the former; in the latter, cells attached firmly to the substratum and formed elongated cytoplasmic processes. Inhibition of cell multiplication by dibutyryl cyclic AMP was related to cell density and to serum concentration. Cells in dibutyryl cyclic AMP-containing media plated at low cell densities multiplied as rapidly as control cells. The final densities cells reached were determined by the serum concentration; in dibutyryl cyclic AMP-containing media these densities were about one-half those of respective control cells. Limitation of cell multiplication by dibutyryl cyclic AMP was reversed by the addition of serum, by resuspending cells at lower densities, or by resuspending cells in media without dibutyryl cyclic AMP. These findings suggested that dibutyryl cyclic AMP may affect the utilization of serum factors by cells. Dibutyryl cyclic AMP did not inactivate serum factors and did not change the rate at which cells depleted the growth medium of serum factors. Dibutyryl cyclic AMP may limit cell multiplication by increasing the cellular requirement for serum factors.     

Induction of refractoriness to thyrotropin stimulation in cultured thyroid cells. Dependence on new protein synthesis.

Cultured dog thyroid cells were used to investigate the mechanism by which previous exposure to thyrotropin (TSH) induces refractoriness to further TSH stimulation of cellular adenosine 3'-5'-monophosphate (cAMP). Refractoriness of the cAMP response to TSH could not be overcome by exposure of the cells to supramaximal stimulatory concentrations of TSH. Although an unknown factor present in human and fetal calf serum was found to inhibit the thyroid cell cAMP response to TSH, this factor could not account for refractoriness because refractoriness could be induced in the absence of serum. Induction of thyroid refractoriness did not appear to be related to cellular concentrations of cyclic AMP, because equal refractoriness was produced by TSH alone or TSH plus the phosphodiesterase inhibitor, 3-isobutyl-1-methyl xanthine. In addition, preincubation of thyroid cells in 10(-4) M cAMP did not result in subsequent refractoriness. Recovery from the refractory process required almost 24 h. Short term (15 min) stimulation with TSH did not produce thyroid cell refractoriness, and reversal of the stimulation was obtained by thorough washing of the cells. Long term TSH stimulation (16 h), however, resulted in both supramaximal cAMP response to TSH, and inclusion of TSH together with cycloheximide did not produce refractoriness. Cyclic AMP phosphodiesterase activity in thyroid cell homogenate was unaltered by TSH or dibutyryl cyclic AMP pretreatment of the cells for up to 24 h, or cycloheximide for up to 4 h. In contrast, TSH-stimulated, but not F--stimulated, adenylate cyclase activity was reduced in thyroid cell homogenates after preincubation of the cells in TSH. Refractoriness to TSH stimulation was not associated with an alteration in the binding of 125I-TSH to cultured thyroid cells. These studies suggest that the thyroid cAMP response to TSH is modulated by an inhibitory mechanism dependent upon new protein synthesis. TSH stimulation itself increases the degree of this inhibition through a mechanism not involving cAMP.     

Effects of calcium ionophore (A-23187) on glucose oxidation and iodide transport in dog thyroid slices.

A calcium ionophore (A-23187, 20 mug/ml) stimulted 14C-1-glucose oxidation in dog thyroid slices to an extent equivalent to that obtained by the optimal concentration of dibutyryl cyclic AMP (1mM). Furthermore, the ionophore augmented the stimulation by dibutyryl cyclic AMP much more than the simple additive effect. The ionophore also enhanced the effect of TSH, but to a lesser extent. Under conditions where organic binding was blocked, T/M ratio of radioiodine concentration was lowered in slices by the ionophore; the findings similar to those obtained with TSH and dibutyryl cyclic AMP. The ionophore exhibited a slightly depressive effect on the basal cyclic AMP level. The elevation by TSH of cyclic AMP levels was also slightly depressed by the ionophore, but statistically insignificant in most cases. These results indicate that calcium ion may play an important role in the TSH regulation of iodide transport and glucose metabolism in the thyroid, in some cases by augmenting the effects of cyclic AMP.     

Regulation of coenzyme A biosynthesis by glucagon and glucocorticoid in adult rat liver parenchymal cells

We studied the effects of glucagon, dibutyryl cyclic AMP and dexamethasone on the rate of [(14)C]pantothenate conversion to CoA in adult rat liver parenchymal cells in primary culture. The presence of 30nm-glucagon increased the rate by about 1.5-fold relative to control cultures (range 1.4-2.3) and 2.4-fold relative to cultures containing 1-3m-i.u. of insulin/ml. The half-maximal effect was obtained at 3nm-glucagon. Dibutyryl cyclic AMP plus theophylline also enhanced the rate by about 1.5-fold. Dexamethasone acted synergistically with glucagon; glucagon at 0.3nm had no effect when added alone, but resulted in a 1.7-fold enhancement when added in the presence of dexamethasone (maximum effect at 50nm). The 1.4-fold enhancement caused by the addition of saturating glucagon concentrations was increased to a 3-fold overall enhancement by the addition of dexamethasone. However, dexamethasone added alone over the range 5nm to 5mum had no effect on the rate of [(14)C]pantothenate conversion to CoA. The stimulatory effect of dibutyryl cyclic AMP plus theophylline was also enhanced by the addition of dexamethasone. Changes in intracellular pantothenate concentration or radioactivity could not account for the stimulatory effects of glucagon, dibutyryl cyclic AMP or dexamethasone. Addition of 18mum-cycloheximide, an inhibitor of protein synthesis, decreased the rate of incorporation of [(14)C]pantothenate into CoA and the enhancement of this rate by glucagon and dibutyryl cyclic AMP plus theophylline in a reversible manner. These results demonstrate an influence of glucagon, dibutyryl cyclic AMP and glucocorticoids on the intracellular mechanism regulating total CoA concentrations in the liver.     

Effects of dibutyryl cyclic AMP on the syntheses of dolichol-linked saccharides and glycoproteins in cultured hepatoma cells. Correlation with the effect on the adhesiveness of the cells.

When the hepatoma cells (AH 70Btc, Clone 10-5) were cultured in the presence of 1 mM-dibutyryl cyclic AMP for 2 days, the incorporation of [14C]glucosamine into protein was increased over 2-fold. At the same time, dibutyryl cyclic AMP increased the incorporation of [14C]glucosamine into dolichol-linked N-acetylglucosamine and NN'-diacetylchitobiose about 1.5-fold and into dolichol-linked oligosaccharides about 3-fold. Analysis of cellular glycoproteins by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after reduction showed that dibutyryl cyclic AMP specifically enhanced the glycosylation of a fibronectin-like glycoprotein with an apparent mol.wt. of 220 000 and two other high-molecular-weight glycoproteins (apparent mol.wts. 270 000 and 185 000). Increased glycosylation of the glycoproteins with mol.wts. of 220 000 and 185 000 was shown to be linked to increased synthesis of the polypeptide portion. In addition to the above effects, dibutyryl cyclic AMP enhanced the adhesiveness of AH 70Btc cells to glass surfaces. Both the effects on the glycosylation pathway and on adhesiveness of cells were reversed by further treatment of the cells with 1 microgram of tunicamycin/ml. The results indicated that dibutyryl cyclic AMP increased the synthesis of dolichol-linked oligosaccharides and N-glycosylation of proteins in AH 70Btc cells. The enhancement of adhesiveness may be mediated by the increased synthesis of dolichol-linked oligosaccharides and also may be related to the increased synthesis of fibronectin.     

Biochemical aspects of cardiac muscle differentiation. Possible control of deoxyribonucleic acid synthesis and cell differentiation by adrenergic innervation and cyclic adenosine 3':5'-monophosphate.

A single injection of either isoproternol or N6, O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) results in an inhibition in the rate of [3H]thymidine incorporation into DNA of differentiating cardiac muscle of the neonatal rat. This inhibition is not due to substantially altered cellular uptake or catabolism of [3H]thymidine. Inhibition of [3H]thymidine incorporation by isoproterenol or dibutyryl cyclic AMP is potentiated by theophylline. Maximal inhibition (95%) is observed 24 h after administration of isoproterenol, and the rate of incorporation returns to a value 80% of control by 72 h. Norepinephrine also inhibits [3H]thymidine incorporation whereas cyclic GMP, N2, 02-Dibutyryl guanosine 3':5'-monophosphate (dibutyryl cyclic GMP), and phenylephrine have little effect. Equilibrium sedimentation analysis of cardiac muscle DNA in neutral and alkaline cesium chloride gradients using bromodeoxyuridine as a density label indicate that isoproterenol and dibutyryl cyclic AMP inhibit [3H]thymidine incorporation into DNA that is replicating semiconservatively. Administration of isoproterenol or dibutyryl cyclic AMP to neonatal rats inhibits by approximately 60% the incorporation of [3H]thymidine into DNA of tissue slices of cardiac muscle prepared 16 h later. [3H]Thymidine incorporation into DNA of tissue slices is into chains that were growing in vivo. This incorporation is linear for at least 4 h of incubation and is inhibited by isoproterenol and dibutyryl cyclic AMP. Inhibition is not due to altered cellular uptake of [3H]thymidine nor is it due to a cytotoxic action. Several other compounds which elevate intracellular levels of cyclic AMP (epinephrine, norepinephrine, glucagon, and prostaglandin E1) also inhibit [3H]thymidine incorporation into DNA or cardiac muscle tissue slices. Cyclic GMP, dibutyryl cyclic GMP, sodium butyrate, and phenylephrine have little effect. Isoproterenol administered together with theophylline to neonatal rats signficantly stimulates the in corporation of [3H]phenylalanine into total cardiac muscle protein and into myosin. This enhanced incorporation may be due in part to an increase in the cellular uptake of [3H]phenylalanine. DNA synthesis decreases progressively in differentiating cardiac muscle of the rat during postnatal development and essentially ceases by the middle of the third week (Claycomb, W. C. (1975) J. Biol. Chem. 250, 3229-3235). In reviewing the literature it was found that this decline in synthetic activity correlates temporally with a progressive increase in tissue concentrations of norepinephrine and cyclic AMP and with the anatomical and physiological development of the adrenergic nerves in this tissue. Because of these facts and data presented in this report it is proposed that cell proliferation and cell differentiation in cardiac muscle may be controlled by adrenergic innervation with norepinephrine and cyclic AMP serving as chemical mediators.     

Chronic and acute effects of thyrotropin on phosphatidylinositol turnover in cultured porcine thyroid cells

The 32P incorporation into phospholipids of isolated porcine thyroid cells, cultured for 1-4 days, has been studied in subsequent 2-h incubations. Along with culture ageing, decreased 32P incorporation into total phospholipid of control cells was observed. The presence of 40 munits/ml TSH during the 2 h incubation yielded a relative increase in labelling of phosphatidylinositol, named 'acute phospholipid effect'. A chronic treatment of the cells with TSH concentration ranging from 0.1 to 10 munits/ml ensured the maintenance of a high turnover rate of total phospholipids. The analysis of individual phospholipids revealed that 1-day culture cells in the presence of 0.1 munits/ml TSH presented a strong increase of phosphatidylinositol labelling. This 'chronic phospholipid effect' of TSH can be reproduced by a chronic treatment with dibutyryl cyclic AMP (10(-3)M) or prostaglandin E2 (10(-6)M), which did not evoke a classical phospholipid effect in a 2 h incubation. If TSH (40 munits/ml) is added to the cells in a 2 h incubation, control cells show the classical phospholipid effect whereas cells chronically treated with TSH, dibutyryl cyclic AMP or prostaglandin E2 presented a 'reverse phospholipid effect' i.e. a relative decrease in phosphatidylinositol labelling. 10(-4)M cycloheximide presence during the last 12 h of culture prevented the establishment of the 'chronic phospholipid effect' and of its consequence, 'the reverse phospholipid effect'. On the basis of these results a scheme is proposed in keeping with current hypotheses concerning phosphatidylinositol metabolism.     

Modulation of nuclear cyclic AMP-dependent protein kinase in dibutyryl cyclic AMP-treated rat H4IIE hepatoma cells.

Biochemical and immunochemical studies were undertaken to quantify the effects of cyclic AMP on cyclic AMP-dependent protein kinase subunit levels in nuclei of H4IIE hepatoma cells. Dibutyryl cyclic AMP (10 microM) caused a significant biphasic (10 and 120 min after stimulation) increase in total nuclear protein kinase activity. The increase observed 10 min after dibutyryl cyclic AMP stimulation was primarily due to an approx. 3-fold increase of catalytic (C) subunit activity, whereas the change observed 120 min after stimulation consisted of an increase in both C subunit and cyclic AMP-independent protein kinase activities. Analysis of nuclear protein extracts by photoaffinity labelling with 8-azido cyclic [32P]AMP identified only the type II regulatory subunit (RII), but not the type I regulatory subunit (RI). Analysis of nuclear RII variants by two-dimensional gel electrophoresis demonstrated that dibutyryl cyclic AMP caused the appearance of two RII variant forms which were not present in the nuclei of unstimulated cells. Using affinity-purified polyclonal antibodies and immunoblotting procedures, we identified an approx. 2-fold increase in the RII and C subunits in nuclear extracts of dibutyryl cyclic AMP-treated hepatoma cells. Finally, the RI, RII and C subunits were quantified by an e.l.i.s.a. which indicated that dibutyryl cyclic AMP increased nuclear RII and C subunits levels biphasically, reaching peak values 10 and 120 min after the initial stimulation. Nuclear RI subunit levels were not affected. These results provide qualitative as well as quantitative evidence for a modulation by cyclic AMP of the nuclear RII and C subunit levels in rat H4IIE hepatoma cells, and indicate a relatively rapid but temporarily limited dibutyryl cyclic AMP-induced translocation of the RII and C subunits to nuclear sites.     

Regulation of phospholipid synthesis inMycobacterium smegmatis by cyclic adenosine monophosphate

Forskolin, an adenylate cyclase activator and a cyclic AMP analogue, dibutyryl cyclic AMP have been used to examine the relationship between intracellular levels of cyclic AMP and lipid synthesis inMycobacterium smegmatis. Total phospholipid content was found to be increased in forskolin grown cells as a result of increased cyclic AMP levels caused by activation of adenylate cyclase. Increased phospholipid content was supported by increased [¹⁴C] acetate incorporation as well as increased activity of glycerol-3-phosphate acyltransferase. Pretreatment of cells with dibutyryl cyclic AMP had similar effects on lipid synthesis. Taking all these observations together it is suggested that lipid synthesis is being controlled by cyclic AMP in mycobacteria.     

Glycogenolytic response to glucagon of cultured fetal hepatocytes. Refractoriness following prior exposure to glucagon.

The glycogenolytic effect of glucagon has been studied in fetal hepatocytes cultured for 3 to 4 days in the presence of cortisol (10 muM). The hepatocytes, when transplanted from young fetuses (15-day-old), contain only minute amounts of glycogen, whereas when cultured 3 to 4 days in the presence of cortisol, they contain high levels of stored glycogen. Glucagon induced a rapid but partial mobilization of glycogen, which was maximal after 2 hours. The half-maximal response was observed with about 0.1 nM glucagon. The glycogenolytic effect of glucagon in fetal hepatocytes is probably mediated by cyclic adenosine 3':5'-monophosphate (cyclic AMP) as in adult liver. This effect was mimicked by cyclic AMP and N-6, O-2-dibutyryl cyclic AMP, (dibutyryl cyclic AMP), and potentiated by theophylline. Glucagon addition was followed by accumulation of cyclic AMP in the cells within 2 min. Glucagon produces a marked stimulation of the rate of glycogen breakdown and an inhibition of the rate of incorporation of [14-C] glucose into glycogen. The glycogeneolytic effect of a single addition of glucagon was reversed within 4 hours. A second addition of glucagon at this time was unable to induce a new glycogenolytic response. A resistance to glucagon stimulation appeared in the cells after a first exposure to the hormone. This refractoriness was also shown by the loss of glucagon-dependent cyclic AMP accumulation and was not linked to the release by the cells of a "hormone antagonist" into the medium. The hepatocytes resistant to the action of glucagon retained their response to cyclic AMP, dibutyryl cyclic AMP, and norepinephrine. Finally, glycogenolytic concentrations of cyclic AMP and of its dibutyryl derivative failed to induce a refractoriness to glucagon.     

Plasminogen activator production by parietal endoderm cells: Stimulation by a protein from pituitary

It has been shown previously that dibutyryl cyclic AMP increases the production of plasminogen activator in mouse parietal endoderm cells. This fact suggested that the production of plasminogen activator by parietal endoderm cells may be under the control of a hormone acting via adenylate cyclase. We have cultured rat parietal endoderm cells in the absence of serum and show that they respond to dibutyryl cyclic AMP with an increase in plasminogen activator production and a change in morphology. We describe the existence of a compound from pituitary which is capable of stimulating plasminogen activator secretion in these cells. Relatively impure preparations of ovine and bovine TSH contain significant amounts of activity, whereas more highly purified preparations of TSH, and all other pituitary hormones tested, are inactive, indicating that the factor is not a known pituitary hormone. The active compound was characterized using ovine and bovine TSH as a source, and it is macromolecular and proteinaceous, and depends on protein synthesis for its effect. The stimulation is enhanced by methylisobutylxanthine, a phosphodiesterase inhibitor, suggesting that the event is mediated by cyclic AMP. This observation leads to the prediction that the coaddition of dibutyryl cAMP and the active compound at nonsaturating concentrations should be additive. Instead, the stimulation is synergistic, and depends on the addition of dibutyryl cyclic AMP first when the compounds are added sequentially. Finally, we show that mouse teratocarcinoma cells chemically induced to differentiate to a cell type indistinguishable from parietal endoderm respond to a source of the compound by increasing plasminogen activator production.     

Bovine thyrotropin inhibits DNA synthesis inversely with stimulation of cyclic AMP production in cultured porcine thyroid follicles

The effects of bovine thyrotropin (TSH) on DNA synthesis and cyclic AMP production were studied in porcine thyroid follicles using suspension culture. During the early 72 hours incubation, the time-dependent uptake of [3H]thymidine by the follicles was observed. In the presence of 10 mU/ml TSH, the uptake of [3H] thymidine was significantly depressed at 72 hours incubation. TSH inhibition of [3H] thymidine incorporation was related to its concentration and the 50% inhibition was observed by using 1.0 mU/ml TSH. Under the same conditions, cyclic AMP production was stimulated by TSH and the stimulation was observed to be related to TSH concentration. In these experiments, the incubation time was 30 min. Dibutyryl cyclic AMP, an analogue of cyclic AMP, inhibited the [3H] thymidine uptake at 72 hours incubation. From these results, it is suggested that TSH inhibits DNA synthesis, and that the inhibition may be mediated by cyclic AMP that is produced by TSH stimulation.     

Uptake and metabolism of 3′,5′-cyclic adenosine monophosphate and N,O′-dibutyryl 3′,5′-cyclic adenosine monophosphate in isolated bovine thyroid cells

1.

1. Accumulation of intracellular radioactivity was measured during incubation of isolated bovine thyroid cells with cyclic [³²P]AMP, cyclic [8-³H]AMP and dibutyryl cyclic [8-³H]AMP. With cyclic [³²P]AMP, ³²P cell/medium ratios ranged from 0 to to 0.04 compared to a maximum ³H cell/medium ratio of 0.29 with cyclic [³H]AMP and 0.16 with dibutyryl cyclic [³H]AMP. The excess of intracellular cyclic [³H] over cyclic [³²P]AMP radioactivity was due to extracellular formation of more penetrable dephosphorylated cyclic AMP metabolites which probably served as precursor of intra-cellular cyclic AMP.     

Biphasic inhibitory and stimulatory effects of thyrotropin on the mitotic activity of thyroid explants cultured in vitro.

An influence of thyrotropin (TSH), dibutyryl cyclic AMP (dbcAMP), triiodothyronine (T3) and thyroxine (T4) on the mitotic incidence in the organ-cultured rat thyroid was studied. The study has revealed biphasic inhibitory and stimulatory effects of TSH on the thyroid mitotic activity. The inhibitory effect occurred 14 h after an exposure of the thyroid explants to TSH and was enhanced by T3. A similar inhibitory effect was observed after an exposure of the explants to dbcAMP. 24 h after the exposure of the thyroid explants to TSH a stimulatory effect on the mitotic incidence was observed. The latter effect was not reproduced by dbcAMP.     

Hyperoxic-induced alterations in lung cell structure and function. Effects on cellular cyclic AMP content and comparison to alterations produced by exogenous cyclic AMP

Hyperoxic exposure in vitro of two lung-derived cell types (the epithelial-derived L2 cells and WI-38 fibroblasts) inhibits cellular replication, produces striking morphologic changes and may result in cell death; these effects have been observed consistently in other cell types. Hyperoxic exposure of L2 cells is associated with an increase in cellular cyclic AMP content (cellular cyclic AMP content 454 +/- 115 fmol/micrograms DNA in cells exposed to pO2 677 Torr for 96 h compared to 136 +/- 17 fmol/microgram DNA in air-grown cells). Hyperoxic exposure of WI-38 fibroblasts is not associated with increased cyclic AMP content. Although cultivation of L2 cells in the presence of exogenous dibutyryl cyclic AMP does inhibit replication and produce morphologic alterations, similar effects are produced by sodium butyrate alone. Hyperoxic exposure alters cyclic AMP metabolism in some cell types, but the structural and functional alterations observed in L2 cells and WI-38 fibroblasts following hyperoxic exposure are not produced by changes in cellular cyclic AMP content.     

Striatal Dopamine Release In Vitro: A β-Adrenoceptor-Regulated Response Not Mediated Through Cyclic AMP

The effects of (-)isoproterenol (10(-6) M), dibutyryl cyclic AMP (10(-3) M), and the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX) (10(-4) M) on in vitro [3H]dopamine ([3H]DA) efflux and synthesis were studied in rat striatal slices continuously superfused with [3H]tyrosine. The beta-adrenoceptor agonist (-)isoproterenol induced an immediate and significant facilitation of [3H]DA efflux but did not alter [3H]DA synthesis as measured by [3H]H2O formation. In contrast, both dibutyryl cyclic AMP and IBMX enhanced [3H]DA synthesis as well as efflux. The presence of IBMX in the superfusing medium did not potentiate the augmentation of [3H]DA efflux caused by (-)isoproterenol. Additionally, the blockade of [3H]DA synthesis by alpha-methyl-p-tyrosine (10(-4) M) completely prevented the action of dibutyryl cyclic AMP on [3H]DA efflux. However, under similar conditions, (-)isoproterenol was still able to increase [3H]DA efflux. The results suggest that (-)isoproterenol can modify striatal DA release through a mechanism not involving cyclic AMP.