Adenosine receptor-mediated changes in cyclic AMP production and DNA synthesis in cultured arterial smooth muscle cells

The effects of adenosine and two analogs, L-phenylisopropyladenosine (L-PIA) and 5'-N-ethylcarboxamidoadenosine (NECA), on cAMP production and on platelet-derived growth factor (PDGF)-stimulated initiation of DNA synthesis in growth-arrested cultures of rat arterial smooth muscle cells (SMC) were studied. The intracellular cAMP concentration was dose-dependently enhanced by micromolar concentrations of adenosine and its analogs, with the potency order NECA greater than adenosine greater than L-PIA. The effect was antagonized, in a competitive manner, by the adenosine receptor antagonist 8-phenyltheophylline (8-PT). The stimulatory effect of adenosine was enhanced by 3 microM dipyridamole an adenosine-uptake blocker. DNA synthesis was inhibited in a parallel manner, showing the same potency order. The inhibition was antagonized by 8-PT. Forskolin, a diterpene with the ability to stimulate the catalytic unit of adenylate cyclase and thereby cAMP formation, potentiated the effects of micromolar concentrations of NECA and L-PIA. Forskolin, by itself, stimulated cAMP production and inhibited DNA synthesis. The forskolin-stimulated increase in cAMP was inhibited by L-PIA at nanomolar concentrations. L-PIA in the nanomolar concentration range also stimulated DNA synthesis when initiation was stimulated with suboptimal concentrations of PDGF. These findings suggest the presence of adenosine receptors of both the A1- and A2-subtype on SM-mediating bidirectional changes of cAMP and DNA synthesis.     

Regulation of thyroid peroxidase activity by thyrotropin, epidermal growth factor and phorbol ester in porcine thyroid follicles cultured in suspension

The activity of thyroid peroxidase (TPO) in porcine follicles cultured for 96 h in suspension with five hormones (5H) still attained over 50% of that in the freshly isolated follicles. On the other hand, the activity in those cultured with 5H + TSH (6H) was several times higher than that cultured with 5H after 96 h, although an initial decrease of TPO activity during the first 24 h of culture was observed in both conditions. The ability of follicles to metabolize iodide (uptake and organification) when cultured with 6H for 96 h was also several times higher than that of those cultured with 5H. The half-maximal dose of TSH for stimulation of TPO activity and iodide metabolism was 0.03-0.04 mU/ml and the effect was mediated by cAMP. These results indicate that in porcine thyroid follicles in primary suspension culture, TPO activity as well as the ability of iodide metabolism is induced by chronic TSH stimulation. In addition, epidermal growth factor (EGF, 10(-9)M) and phorbol 12-myristate 13-acetate (PMA, 10(-8) M) completely inhibited TSH stimulation on both activities and also basal (5H) activity of iodide metabolism.     

Noninactivation of thyrotropin by cultured porcine thyroid cells.

Freshly isolated porcine thyroid cells were cultured in the presence of highly purified porcine thyrotropin. Cells associate into follicles between the second and tenth day of culture and later form a monolayer. The biological and immunological activity of thyrotropin was measured daily in the media. Thyrotropin concentration and biological activity remained unchanged from the onset of the culture up to day 14. Limiting factors influencing thyroglobulin biosynthesis do not appear before day 13. The loss of follicular organization at day 10 cannot be explained by thyrotropin degradation in the medium. Considering the number of receptors per cell and the half life of the thyrotropin . receptor complex in the two dissociation compartments previously demonstrated, it appears in terms of both biological activity and affinity for the receptors that the thyrotropin molecules released from the first compartment do not differ from native molecules. It can be calculated that at least 31% of the molecules released from the second compartment are not inactivated. Thus, it is probable that the catabolism of thyrotropin on the receptor, or near the receptor site, does not play an important role in the regulation of thyroid cell function in vitro.     

Changes in cyclic AMP level of rat thyroid by acute and chronic stimulation of thyrotropin in vivo.

To assess a possible postmortem change in the level of cyclic AMP, the thyroids were snap-frozen at various time intervals after removal. Rats were fed a low-iodine diet (LID) with PTU for 2 weeks and a week after PTU discontinuation (PTU withdrawal). In all cases, the cyclic AMP level tended to increase as time elapsed from removing till fixing the thyroids, but in the PTU withdrawal group, the level was rapidly increased 2-fold after 5 min. In an acute experiment, the thyroids were removed under anesthesia and frozen rapidly. Intravenous administration of ovine thyrotropin (250 mU) and TRH (500 ng) brought about a rapid increase in the thyroidal level of cyclic AMP to 40% to 20% over the control level. Two weeks after PTU treatment, circulating thyrotropin was increased to a maximum of 19-fold and a further enhancement ("rebound") was observed after PTU withdrawal. PTU treatment led to an increase in the thyroidal level of cyclis AMP pre mug DNA to 60% over the control value. Following PTU withdrawal, the rise in the level of cyclic AMP returned to the normal level. However, there was no change in the concentration when it was expressed as per mg wet tissue weight. Therefore, the increase in the thyroidal concentration of cyclic AMP per mug of DNA may be due to an increase in volume of the follicular cells.     

Effect of gelatin on the cyclic AMP response of primocultured hog thyroid cells to acute thyrotropin stimulation

The cyclic AMP response of cultured hog thyroid cells to acute thyrotropin stimulation was shown to be under a dual regulatory control by thyrotropin: both positive and negative regulation have been described. When added to the culture medium, gelatin (0.25%) promoted the reorganization of the cells into follicle-like structures, as does thyrotropin. Unlike thyrotropin, gelatin did not induce an increase in intracellular cyclic AMP but enhanced the acute cyclic AMP response to thyrotropin in cells cultured in gelatin-containing medium. When both gelatin and thyrotropin were present, the positive effect of low concentrations of hormone (less than 50 μU/ml) was increased whereas the refractory process observed in the presence of higher concentrations of hormone (greater than 50 μU/ml) was unchanged. These effects of gelatin might be mediated by interaction of the denatured collagen molecules with external proteins of the plasma membrane of thyroid cells.     

Phorbol esters modulate cyclic AMP accumulation in porcine thyroid cells

In cultured porcine thyroid cells, during 60 min incubation phorbol 12-myristate 13-acetate (PMA) had no effect on basal cyclic AMP accumulation and slightly stimulated cyclic AMP accumulation evoked by thyroid stimulating hormone (TSH) or forskolin. Cholera toxin-induced cyclic AMP accumulation was significantly stimulated by PMA. On the other hand, cyclic AMP accumulation evoked by prostaglandin E1 or E2 (PGE1 or PGE2) was markedly depressed by simultaneous addition of PMA. These opposing effects of PMA on cyclic AMP accumulation evoked by PGE and cholera toxin were observed in a dose-related fashion, with half-maximal effect of around 10(-9) M in either case. The almost same effects of PMA on cyclic AMP accumulation in basal and stimulated conditions were also observed in freshly prepared thyroid cells. The present study was performed in the presence of phosphodiesterase inhibitor, 3-iso-butyl-1-methylxanthine (IBMX), indicating that PMA affected adenylate cyclase activity. Therefore, it is suggested that PMA may modulate the production of cyclic AMP in response to different stimuli, possibly by affecting several sites in the adenylate cyclase complex in thyroid cells.     

Effect of gelatin on the cyclic AMP response of primocultured hog thyroid cells to acute thyrotropin stimulation.

The cyclic AMP response of cultured hog thyroid cells to acute thyrotropin stimulation was shown to be under a dual regulatory control by thyrotropin: both positive and negative regulation have been described. When added to the culture medium, gelatin (0.25%) promoted the reorganization of the cells into folicle-like structures, as does thyrotropin. Unlike thyrotropin, gelatin did not induce an increase in intracellular cyclic AMP but enhanced the acute cyclic AMP response to thyrotropin in cells cultured in gelatin-containing medium. When both gelatin and thyrotropin were present, the positive effect of low concentrations of hormone (less than 50 microU/ml) was increased whereas the refractory process observed in the presence of higher concentrations of hormone (greater than 50 microU/ml) was unchanged. These effects of gelatin might be mediated by interaction of the denatured collagen molecules with external proteins of the plasma membrane of thyroid cells.     

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.     

Stimulation by thyrotropin and cyclic AMP of the proliferation of quiescent canine thyroid cells cultured in a defined medium containing insulin

We have developed serum-free primary cultures of differentiated follicular dog thyroid cells which allow the study of the hormonal control of cell proliferation. The cooperation of insulin and increasing cellular cyclic AMP by thyrotropin triggers the DNA synthesis and the proliferation. Dog thyroid cells are an example of a system in which cyclic AMP is a sufficient signal to stimulate the proliferation in quiescent cells.     

Exposure of cultured hepatocytes to cyclic AMP enhances the vasopressin-mediated stimulation of inositol phosphate production.

Isolated rat hepatocytes in primary monolayer culture were maintained for 18-24 h in the presence of 10% (v/v) serum and [3H]inositol. Vasopressin (100 nM) stimulated the production of inositol mono-, bis- and tris-phosphates (IP1, IP2, and IP3). Prior exposure of hepatocytes to 8-bromo cyclic AMP (8Br-cAMP; 100 microM), but not 8-bromo cyclic GMP, enhanced the vasopressin-mediated stimulation of inositol phosphate accumulation, but had no significant effect on their formation in the absence of vasopressin. The effect of the cyclic AMP analogue was mimicked by glucagon (10 nM), and was seen whether cyclic AMP or glucagon was added 5 min or 12 h before the addition of vasopressin. An 8 h incubation with dexamethasone (100 nM) enhanced the accumulation of IP3, but not that of IP2 or IP1, in the presence of 8Br-cAMP and vasopressin. Cycloheximide or actinomycin D had little effect on the vasopressin stimulation of inositol phosphate accumulation, after an 8 h incubation in the presence or absence of 8Br-cAMP.     

Release of an endothelial cell growth factor from cultured porcine thyroid follicles

It has been proposed from in vivo studies that thyroid angiogenesis during thyroid enlargement may be due to paracrine mitogenic factors released by epithelial thyroid cells. To study this paracrine growth regulating communication between thyroid cells and endothelial cells in vitro, culture medium from isolated porcine thyroid follicles was investigated for a growth promoting effect on porcine aortal endothelial cells. Serum-free conditioned medium (CM) from thyroid follicles in suspension culture contains a dose-related mitogenic activity which stimulates endothelial cell growth up to 197%. Stimulation of the thyroid follicles with TSH (1 mU/ml) significantly reduced the mitogenic activity for endothelial cells in CM to 131%. Thyroid hormones had no influence on mitogenic activity in CM. When follicles were treated with iodide (20 microM) during CM production, no proliferation of endothelial cells was observed by this CM. In contrast, CM from epidermal growth factor-treated thyroid follicles significantly enhanced the mitogenic activity for endothelial cells up to 235%. The mitogenic activity was precipitable by saturated ammonium sulfate, showed high affinity to heparin by chromatography on heparin-sepharose, and was abolished after treatment of CM with trypsin. On gel electrophoresis the heparin-binding fraction showed a double band with a mol wt of 15 and 15.5 k. These data show a paracrine mitogenic activity on endothelial cells released by thyroid follicles which is regulated by TSH, epidermal growth factor, and iodide in parallel with the direct effect of these substances on thyroid cell growth. The data suggest that the mitogenic factor is a polypeptide, which belongs to the heparin-binding growth factors.     

Prevention by nicotinamide of desensitization to thyrotropin stimulation in cultured human thyroid cells

The presence of 50 mM nicotinamide together with 100 milliunits/ml of TSH in the incubation medium prevented the decline in human thyroid cell cAMP from maximum, stimulated levels (15-30 min) that occurs when the cells are exposed to TSH alone. Nicotinamide in the absence of TSH did not increase thyroid cell cAMP content. TSH desensitization, and its prevention by nicotinamide, occurred in the presence or absence of 3-isobutyl-methylxanthine. 1-Methyl nicotinamide and N'-methyl nicotinamide similarly prevented TSH desensitization. Recovery from TSH desensitization was prolonged and incomplete after 72 h. The presence of 50 mM nicotinamide hastened recovery from desensitization. Desensitization of the cAMP response to 10(6) M prostaglandin E1 and 1 mM adenosine was unaffected by nicotinamide. Other inhibitors of poly(ADP-ribose) polymerase activity, 5-bromouridine, 5-bromo-2'-deoxyuridine, and thymidine (all at 50 mM) completely or partially prevented TSH desensitization. Pyridoxine (50 mM) similarly prevented this phenomenon. As with dog thyroid cells, 10(-4) M cycloheximide blocked TSH desensitization. The combination of 10(-4) M cycloheximide and 50 mM nicotinamide had a synergistic effect in augmenting the thyroid cell cAMP response to TSH stimulation.     

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.     

Potentiation of cholera toxin-stimulated cyclic AMP production in cultured cells by inhibitors of RNA and protein synthesis

Cyclic AMP increased 8- to 10-fold after a 3-h treatment with 6 nM cholera toxin in rat C6-2B astrocytoma cells. In the presence of cycloheximide, cholera toxin increased intracellular cyclic AMP about 50-fold. Qualitatively similar potentiation of cholera toxin action by cycloheximide was observed in isolated swine aortic vascular smooth muscle cells. Cycloheximide, by itself, had no effect upon cyclic AMP levels and did not alter the apparent Ka for cyclic AMP generation by cholera toxin in the cells. Also, cycloheximide did not appear to augment cholera toxin action via inhibition of cyclic nucleotide phosphodiesterase. Puromycin and actinomycin D also augmented cholera toxin action in C6-2B cells. Potentiation of cholera toxin-increased cyclic AMP formation by cycloheximide was correlated with the inhibition of [14C]leucine incorporation into protein. These results indicate that the ability of cholera toxin to stimulate cyclic AMP production in C6-2B astrocytoma and swine vascular smooth muscle cells is enhanced by inhibition of de novo protein synthesis.     

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.     

Thyrotropin and cyclic AMP regulation of ras proto-oncogene expression in cultured thyroid cells

We examined the effect of thyrotropin (TSH) on intracellular levels of c-ras mRNA in a line of differentiated rat thyroid cells obtained from normal Fischer rat thyroids. These cells are totally dependent on TSH for growth. TSH stimulation of quiescent cells increased c-ras mRNA content, with a maximal response (730% of basal) after 6 h, and a decline towards basal levels after 24 h. Dibutyryl cAMP and forskolin mimicked this stimulatory effect of TSH on c-ras, but did not enhance beta-actin mRNA content. This study demonstrates hormonal and cyclic nucleotide control of c-ras expression in a well-differentiated, non-tumorogenic mammalian cell.     

Transient increase in prostaglandin production as an acute response of thyroid isolated follicles to thyrotropin

1. Isolated follicles obtained from pig thyroid were incubated in Krebs-Ringer bicarbonate buffer containing glucose. Following an immediate increase, immunoreactive prostaglandin E in the reaction system reached a plateau. When 80 mU/ml of thyrotropin was added 30 min after the onset of incubation, the prostaglandins abruptly increased within 1 min and returned to the original level by 10 min, while no appreciable change was observed in the absence of TSH. 2. The minimal effective dose of TSH for this induction of prostaglandin increase was between 1 and 5 mU/ml under the present conditions. 3. The specificity of the stimulant was tested by using the same number of units of two thyrotropin preparations and the same weight of other hormones. ACTH was not active. Lutropin and follitropin were considerably less active than, and epinephrine was as active as, thyrotropin. 4. The phenomenon may reflect the TSH-induced transient accumulation of diacylglycerol (B.B.R.C. 97, 759 and 765) as a possible source of prostaglandins.