Stimulation by forskolin of the thyroid adenylate cyclase, cyclic AMP accumulation and iodine metabolism

Forskolin, a diterpene hypotensive drug, activates adenylate cyclase in brain and in some other tissues (Seamon et al., 1981). Forskolin activated adenylate cyclase in particulate preparations and enhanced cyclic AMP accumulation in slices of dog thyroid. These effects were maximal within minutes and remained constant afterwards. The action of forskolin on intact cells disappeared rapidly after washing. It reproduced two known cyclic AMP-mediated TSH effects: the activation of secretion and of protein iodination. Forskolin thus provides a very convenient tool for the study of the action of defined elevations of cyclic AMP level in thyroid cells. The activation by forskolin of adenylate cyclase was not reduced by Mn2+ which uncouples TSH and PGE1 action. This suggests that in the thyroid also, forskolin acts beyond the receptor level. The effect of forskolin on cyclic AMP accumulation was inhibited by the known negative regulators of this system in the thyroid, acetylcholine, iodide, norepinephrine, PGF1 alpha and adenosine. On the other hand, forskolin potentiated the effects of TSH, PGE1 and cholera toxin. These data show that, though it does not require the receptors for its action, forskolin does not uncouple them from the catalytic unit of adenylate cyclase.     

Further characterization of the iodide inhibitory effect on the cyclic AMP system in dog thyroid slices

Iodide inhibits cyclic AMP accumulation in the thyroid by a process which is prevented by inhibition of iodide uptake and of thyroid peroxidase. By a similar process, it also exerts other independent effects such as the enhancement of iodinated protein release. Iodide inhibited the stimulation of adenylate cyclase by prostaglandin E₁, cholera toxin and forskolin. The action of iodide was not relieved by phosphodiesterase inhibitors and was not additive with the effect of norepinephrine or adenosine. Iodide did not decrease the cellular level of ATP. The data are compatible with an inhibition of adenylate cyclase beyond the level of the receptor, presumably at the level of the catalytic unit or its interaction with the positive transducing unit N_s. The effect of iodide required TSH for its expression but not for its installation. It was decreased under all conditions in which iodide organification was decreased: decreased iodide or increased methimazole concentration, absence of calcium in the medium, etc. However, the relation between iodide binding to proteins and effect was not linear. The effect was not relieved by washing in the absence of iodide and in the presence of perchlorate, but it was partly reversible in the presence of methimazole propylthiouracyl or thiourea. It was not relieved by cooling to 20°C and cytochalasin b, which block stimulated thyroglobulin hydrolysis and iodothyronine release, nor by actinomycin D, cycloheximide, puromycin, mepacrine or indomethacin. The data suggest that iodide binds to a saturable cell component by a reaction which is reversible only in the presence of thiol-containing drugs.     

Studies on the mechanism of desensitization of the cyclic AMP response to TSH stimulation in a cloned rat thyroid cell line

We examined aspects of the mechanism of desensitization of adenylate cyclase activation by TSH in a cloned line of rat thyroid cells (FRTL). Increasing FRTL intracellular cAMP concentrations by preincubation for 6 h in either 1 mM dBcAMP or 100 microM forskolin did not induce TSH desensitization. Forskolin stimulation was unimpaired in TSH-desensitized cells, indicating 'uncoupling' of the adenylate cyclase catalytic unit from the TSH receptor. Stimulation by the Ni inhibitory pathway of the adenylate cyclase by epinephrine (10(-6) M-10(-4) M in the presence of 10(-4) M propranolol) was unaltered in cells previously desensitized to TSH. That is, Ni-mediated inhibition of adenylate cyclase was additive to TSH desensitization. Pre-exposure of FRTL cells for 18 h to 50 ng/ml pertussis toxin did not prevent the induction of TSH desensitization. TSH desensitization was prevented by cycloheximide or actinomycin D added during the last 3-4 h of a 6 h period of TSH stimulation. The rates of turnover of the putative desensitization protein and its mRNA therefore appear to be similar.     

Preparation of porcine thyroid follicles with preserved polarity: functional and morphological properties in comparison to inside-out follicles

One of the main problems in establishing isolated thyroid follicles in vitro is their tendency to form inside-out follicles. The reason for this change in polarity is unknown. We describe here a method for the preparation of stable thyroid follicles with preserved polarity for at least 6 days. Isolated follicles were obtained by infusion of collagenase (1.5 mg/ml) dissolved in minimal essential medium into the artery of intact thyroid glands. The morphological and functional properties of these follicles were compared to inside-out follicles. These inside-out follicles were obtained by digestion of minced thyroid tissue in a collagenase (1 mg/ml) solution. The polarity of follicles was proved by morphological criteria. Follicles with preserved polarity did not change polarity for at least 6 days in the presence of 1% or 5% fetal calf serum. As the culture conditions for both preparation methods were identical, we conclude that the preparation method rather than the culture condition is responsible for the preservation of cell polarity of isolated thyroid follicles in our system. Increases in cyclic AMP levels induced either by bovine thyrotropin (10(-3) U/ml) or by isoproterenol (10(-6) M) as well as iodide uptake and organification were rapid and significant in right-side-right follicles but not in inside-out follicles. Therefore the TSH receptor and the beta-adrenergic receptor appear to be exclusively located at the basal membrane of follicular cells. In addition, iodide uptake apparently is unidirectional.     

Thyroxine secretion by isolated hog thyroid cells: a cyclic AMP independent pathway.

The release of 131I-labeled thyroxine (T4) from isolated hog thyroid cells was increased 1.5--2-fold by thyrotropin (TSH). Dibutyryl cyclic AMP failed to reproduce this TSH action. In this in vitro system another cell activity, T4 synthesis, was stimulated in an essentially identical fashion by TSH and dibutyryl cyclic AMP (time course of action, dose-response relationship). 3-Isobutyl-1-methylxanthine (IBMX), 0.5 mM, did not alter the basal [131I]T4 release whereas it enhanced the [131I]T4 synthesis. TSH, 60 MU/ml, increased the intracellular cyclic AMP concentration 3-4-fold. Chlorpromazine (5 X 10(-4)M) abolished the TSH stimulation of cyclic AMP accumulation but did not alter the TSH-induced increase in [131I]T4 secretion. It is concluded that the TSH action on [131I]T4 secretion by isolated thyroid cells is not mediated by the adenylate cyclase-cylic AMP system.     

Forskolin effects on the cAMP system and steroidogenesis in the immature rat ovary

The diterpene forskolin was found to activate the adenylate cyclase system in intact tissue and membrane preparations of the immature rat ovary. The cyclic AMP (cAMP) response reached a maximal level after 5 min and no decline was observed even after 4 h of incubation. Forskolin stimulated production of both progesterone and testosterone in a pattern similar to that produced by luteinizing hormone (LH) or dibutyryl-cAMP (dbcAMP). In combination with LH, follicle-stimulating hormone (FSH) or prostaglandin E2 (PGE2), forskolin potentiated the hormone effects on adenylate cyclase activity in membrane preparations. Pretreatment with LH or PGE2 desensitized the cells to further hormone stimulation, while the forskolin response was unaffected. Pre-exposure to forskolin did not desensitize the cells to a subsequent stimulation by LH or PGE2. The presence of 8-bromo-cAMP (brcAMP) in the preincubation medium reduced the subsequent hormone response. These results demonstrate a rapid and sustained activation of the adenylate cyclase system by forskolin in the rat ovary. The steroidogenic response was similar to that of known stimulators of ovarian cells (LH, dbcAMP). The inability of forskolin to induce desensitization of the adenylate cyclase system demonstrates, however, important differences between hormone and non-hormone activation. Consequently, forskolin can be a useful tool for investigation of the mechanisms involved in the desensitization process.     

Thyrotropin increases 5'-nucleotidase activity in primary cultures of porcine thyroid cells

Thyrotropin given to support-anchored cultures of porcine thyroid cells, either in a serum-containing or in a serum-free system, produced an increase of about 50% in the total activity of 5'-nucleotidase. In the serum-free culture, in which TSH was administered to well-reformed follicles, this increase in 5'-nucleotidase activity concerns both the ecto-enzymic and intracellular forms of the enzyme and it coincides with the period of several days during which several glycosyltransferase activities are elevated and thyroglobulin production increased. Taken together, and in view of a recent in vitro study (Brandan and Fleisher, 1982) documenting the fate of uridine diphosphate in Golgi vesicles, these results suggest that there might be a functional correlation between the stimulation of 5'-nucleotidase and an increased production of nucleoside mono- and diphosphates when the activity of a number of glycosyltransferases is increased.     

Effect of thyrotropin and of LATS on mouse thyroid cyclic AMP

The effects of thyrotropin and of LATS on the concentration of cyclic AMP in the mouse thyroid were studied. Increase was readily effected with 2–50 mU thyrotropin/ml in vitro, in the presence of 10 mM theophylline. By i.v. injection 2, 10, and 50 mU progressively increased cyclic AMP concentration; responses to 10 and 50 mU were evident within 3 min. After i.v. injection of thyrotropin, incubating thyroid lobes for 10 min in buffer that contained theophylline resulted in even greater concentrations of the nucleotide. With smaller doses (e.g., 2 or 10 mU) the in vivo effect on the concentration of cyclic AMP seemed largely to be masked by hydrolysis of the nucleotide. LATS-IgG was only minimally effective in vitro in increasing the concentration of cyclic AMP with 10 min of incubation; 90-min incubation led to more clear-cut increases. Injection i.v. of LATS (2000% in bioassay), followed in 5 min by 10-min incubation in buffer that contained theophylline, enhanced twofold the concentration of the nucleotide. There was also a measurable increase in thyroids removed 2 or 7 hr (but not at 23 hr) after i.p. injection of LATS-IgG.     

Studies on the control and dynamics of thyrotropin secretion from isolated adenohypophyseal cells.

Thyrotropin (TSH) secretion from isolated anterior pituitary cells has been studied using the technique of cell column perifusion. The consistency in secretory rate and temporal profiles of TSH output in response to stimulation illustrated that the system is suitable for studying the kinetics of stimulation and inhibition of secretion. During perfusion TSH release was stimulated in response to a variety of secretogogues, namely TRH, raised potassium concentrations and phosphodiesterase inhibitors. The onset and termination of the secretory responses were rapid and displayed a temporally biphasic pattern of secretion. Dose-related increases in TSH output in response to TRH and consistent responses to repetitive pulses of TRH (5.5 X 10-10 M) during a 4 h period were demonstrated. Studies on the dynamics of thyroid hormone feedback on TRH-stimulated TSH secretion indicated that inhibition was manifest within 1 h and reached a maximum after 2 1/2 h during continual exposure to thyroid hormones. Isobutylmethylxanthine (IBMX) potentiated the effect of raised K+ as well as that of TRH on TSH secretion, suggesting an as yet unidentified relationship between Ca2+ and cyclic AMP.     

Studies on the role of calcium and cyclic nucleotides in the control of TSH secretion.

The role of putative mediators in the control of thyrotropin (TSH) secretion has been investigated by monitoring hormone release from isolated anterior cells in response to agents and conditions which modify cyclic nucleotide concentrations or calcium fluxes. The secretory response to 5-min pulses of TRH, raised K⁺ concentration and A-23187 was diminished within 5 min when Ca²⁺-free perifusion was begun 80 sec prior to the pulse. In contrast, the response to theophylline and IBMX was unaffected under these conditions. Both IBMX and dibutyryl cyclic AMP potentiated the effects of TRH and raised K⁺concentration but not that of A-23187. Methoxyverapamil inhibited TSH secretion stimulated by TRH, raised K⁺ concentration and IBMX but not that induced by A-23187. Calcium efflux showed a similar temporal profile to hormone secretion in response to TRH, IBMX and raised K⁺ concentration. It is proposed that one interpretation of these findings is that there is an interrelationship between calcium ions and the cyclic nucleotides in the control of TSH secretion and that cyclic nucleotides may act at the level of the Ca²⁺ channel to modulate Ca²⁺ entry.     

Evidence of a Stimulatory Effect of Cyclic AMP on Pancreatic Lipase and Colipase Synthesis in Rats

The effects of endogenous and exogenous cyclic AMP on the synthesis of pancreatic lipase, colipase, and amylase were studied. Pancreatic lobules were prepared and incubated with forskolin, dibutyryl cyclic AMP (dbcAMP), and dibutyryl cyclic GMP (dbcGMP), respectively, in the presence of “S-cysteine”. The individual pancreatic enzymes were isolated by polyacrylamide gel electrophoresis, and the incorporation of radioactive cysteine into lipase, colipase, and amylase was determined. Incubation with forskolin (25 uM) rapidly increased lipase synthesis rate within 30 min, followed by an increase in colipase synthesis rate after 60 min of incubation. Amylase synthesis rate did not change during the 1st h of incubation but decreased slightly when incubated for 2h. Incubation of pancreatic lobules with dbcAMP (1 mM) for 1 h also stimulated the incorporation of cysteine into lipase and colipase by 21% and 25%, respectively, whereas incubation with dbcGMP had no effect on the synthesis rates of lipase and colipase. Neither dbcAMP nor dbcGMP had any effect on synthesis rate of amylase. It is concluded that cyclic AMP might be an important intracellular signal for the synthesis of pancreatic lipase and colipase in the rat     

Responsiveness of glycosyltransferases to thyrotropin in a serum-free culture of porcine thyroid cells

Administration of thyrotropin to porcine thyroid follicles, obtained in a serum-free chemically defined medium, provoked marked increases in the activities of several glycosyltransferases involved in protein N-glycosylation. The coincidence of these effects with a previously demonstrated enhancement of thyroglobulin production renders a relationship between these events likely. The most important stimulation was for peptide oligosaccharyltransferase (3-fold). Among the enzymes involved in the synthesis of the lipid oligosaccharide donor, Dol-P glycosyl- and mannosyltransferases were increased 1.5-fold, and Dol-P N-acetylglucosaminylphosphotransferase only 1.15-fold. As regards terminal glycosyltransferases, asialofetuin sialyltransferase was increased 2-fold and ovomucoid galactosyltransferase only 1.2-fold. There was a continuous release of the latter two enzymes into the culture medium.     

Active transport of iodide in isolated porcine thyroid cells. Application to an in vitro bioassay of thyrotropin.

Porcine thyroid cells were cultured for 2 days with or without dibutyryl cyclic AMP or thyrotropin (TSH). Then they were isolated post-culture by a gentle treatment with a calcium chelating agent. Some characteristics of the iodide transport system were studied in these thyroid cell suspensions.Iodide influx is a saturable, temperature- and energy-dependent phenomenon. It is blocked by ouabaïn, N-ethylmaleimide, dinitrophenol, cardiotoxin, low Na⁺ concentration and harmaline. Only 3% of the intracellular iodide is trichloracetic-acid-insoluble at equilibrium. The apparent Michaelis constant (K_m) of the transport system is 30 μM.For monolayer cells, the decrease of C/M ratio, increase of apparent K_m, and decrease of V_max between day 0 (freshly isolated cells) and day 6, indicate a loss of iodide-trapping ability up to passive diffusion. To the contrary, high values of C/M and normal K_m (30 μM) are observed in TSH follicles from reassociated cells.At iodide equilibrium, thryotropin, prostaglandins E₁ and E₂ and long-acting thyroid stimulator (LATS), induce a fast release of iodide. This release is dose-dependent in the first 5 min. It has been used to develop a bioassay of TSH and a fast detection of LATS.     

Stimulation and inhibition by LHRH analogues of cultured rat Leydig cell function and lack of effect on mouse Leydig cells

The effect of 2 luteinizing hormone-releasing hormone (LHRH) analogues (10^?8–10^?6 M) on the functional activity (testosterone and cyclic AMP production and [¹²⁵I]hCG binding) of purified mouse Leydig cells in culture was examined. The analogues were found to have no significant effect on the cells over a period of 3 days. No specific binding of a labelled analogue to impure or pure mouse Leydig cells could be detected. In contrast high levels of specific binding to impure rat interstitial cells occurred. Centrifugation of the rat interstitial cells on 0–90% Percoll gradients showed that the LHRH analogue bound specifically to the active lutropin-responsive Leydig cells.The purified rat Leydig cells were cultured in the presence of LHRH analogue (ICI 118630) (10^?7 M) and after an initial lag period (2 h) a marked stimulation of testosterone production occurred over a 32-h period (up to 400 ng/10⁶ cells). The response to LH alone increased with time in culture up to 10 h, and the LHRH analogue enhanced this LH-stimulated testosterone production. When the cells were cultured for longer time periods (24 h) the LHRH analogue was found to inhibit LH-stimulated testosterone production at all concentrations of LH used (P < 0.01). The LHRH analogue had no consistent effect on LH-stimulated cyclic AMP production, although when added alone, cyclic AMP production was increased.These results show that LHRH analogues do not bind to or have any detectable effect on mouse Leydig cells in vitro. However, LHRH analogue does bind specifically to purified rat Leydig cells. After a short lag period the analogue stimulates testosterone production which turns to inhibition after 20 h in culture.     

PGE2 binding sites and PG-stimulated cyclic AMP accumulation in rat isolated glomeruli and glomerular cultured cells

[3H]PGE2 specifically bound to isolated glomeruli. The KD value and the number of sites were 80 nM and 528 fmoles/mg respectively. PGE1 and PGE2 resulted in equipotent inhibition of binding whereas PGI2 was markedly less active. It was not possible to demonstrate specific receptors for PGE2 in glomerular mesangial and epithelial cultured cells. PGE1, PGE2 and PGI2 (0.1-100 microM) stimulated cyclic AMP concentration both in isolated glomeruli and glomerular cultured cells. Basal cyclic AMP in epithelial cells was greater than in mesangial cells or glomeruli. The cyclic AMP accumulation in the presence of PGs was greatest in mesangial cells. Maximum stimulation was in the range 300-1400%. For the three preparations, PGE2 and PGE1 produced a greater effect than PGI2. ED50 values were identical for PGE1 and PGE2 (5 microM for epithelial cells and glomeruli, 20 microM for mesangial cells). ED50 value for PGI2 were lower than those for PGE1 or PGE2 (0.2, 2 and 5 microM for glomeruli, epithelial cells and mesangial cells, respectively). The effects of the three PGs were not additive when tested at maximally effective concentrations. These results demonstrate that PGE1, PGE2 and PGI2 stimulate glomerular and cellular cyclic AMP. A relationship between [3H]PGE2 binding sites and this biological effect has not been established. The physiological events secondary to the increase in glomerular cyclic AMP are also yet to be determined.     

Influence of calcium, parathyroid hormone and ionophore a-23187 on cyclic nucleotide concentrations of isolated renal tubules

Effects of parathyroid hormone (PTH) and the divalent cation ionophore A-23187 on concentrations of cyclic AMP (cAMP) and cyclic GMP (cGMP) were investigated in isolated renal tubules from male hamsters. Both PTH and A-23187 increased cGMP concentrations, effects which required the presence of extracellular calcium. Increases in cGMP in response to both agents were proportionate to concentrations of calcium between 0.01 and 1.0 mM.Pretreatment of tubules with ionophore prevented any further increase in cGMP in response to PTH. Increases in cAMP in response to PTH were not altered by extracellular calcium, while smaller increases due to A-23187 were completely calcium-dependent. A-23187 increased efflux of ⁴⁵Ca from renal tubules and resulted in decreased accumulation and net uptake of ⁴⁵Ca at low extracellular calcium concentrations (2–3 μM). Increasing extracellular calcium (0.1–3 mM) proportionately reversed the inhibition in ⁴⁵Ca accumulation and resulted in increases in cGMP levels which were directly related to increases in ⁴⁵Ca accumulation. The results indicate that changes in cGMP concentrations of isolated renal tubules following either PTH or A-23187 are related to changes in intracellular calcium.     

Steroidogenesis and extracellular cAMP accumulation in adrenal tumor cell cultures.

ACTH stimulated steroidogenesis and cAMP (adenosine 3',5'-monophosphate) accumulation in an adrenocortical mouse tumor cell line (clone Y1) with Kd values which differed by more than one order of magnitude (5.2 X 10(-11) M and 7 X 10(-10) M, respectively). All of the cAMP formed in response to added ACTH appeared extracellularly in 5- or 30-min incubations. ACTH, at 5 and 10 muU/ml, stimulated steroidogenesis to 25% and 40% of maximum activity; and increased the extracellular accumulation of cAMP 1.4-fold and 2.3-fold, respectively. The effects of ACTH appeared to be via an action on intracellular ATP, specific for cAMP and dependent on an ACTH-sensitive adenylate cyclase system. These observations indicate that ACTH increases cAMP accumulation in Y1 cells at virtually all steroidogenic concentrations and suggest that cAMP is an essential component of ACTH-stimulated steroidogenesis.     

Effect of misoprostol on histamine-stimulated acid secretion and cyclic AMP formation in isolated canine parietal cells

Isolated canine parietal cells were used to study the ability of misoprostol to inhibit acid secretion in the presence of a number of acid secretagogues. Misoprostol inhibited histamine-stimulated acid secretion in a dose-dependent and noncompetitive manner. A concentration of 2–3×10^–9 M misoprostol inhibited maximal histamine-stimulated acid secretion by one half. Misoprostol had little to no effect on acid secretion stimulated by carbachol and dibutyryl cAMP, had no effect on the acid secretion directly stimulated by pentagastrin, and only modestly inhibited acid secretion stimulated by forskolin. Misoprostol noncompetitively inhibited cAMP formation in response to histamine, with an IC₅₀ value similar to that for the inhibition of histamine-stimulated acid secretion. These results indicate that: (1) misoprostol specifically inhibits histamine-stimulated acid secretion in parietal cells, and (2) the antisecretory action of misoprostol is closely related to the reduction of histamine-stimulated cAMP formation with the site of major action most likely in the coupling process between histamine H₂ receptor sites and histamine-sensitive adenylate cyclase.     

自主性功能性甲状腺腺瘤TSH受体基因突变研究

目的　研究TSH受体 (TSHR)基因突变在自主性功能性甲状腺腺瘤 (AFTA)发病中的作用。方法　以 14例AFTA作为研究对象 ,另 4例为毒性多结节性甲状腺肿、7例扫描呈“冷结节”的甲状腺腺瘤及AFTA周围正常甲状腺组织作为对照 ,酚 氯仿 异戊醇法提取基因组DNA ,对目的基因片段进行聚合酶链反应—单链构象多态性 (PCR SSCP)分析及DNA序列分析。结果　在 14例自主性功能性甲状腺瘤标本中 ,SSCP检测出 6例条带变异的个体 (4 3 % ) ,对其中 3例进行DNA序列测定 ,发现 1例为 6 2 0位密码子的点突变 ,苏氨酸被脯氨酸置换 (T6 2 0P ,ACC→CCC)。 2例为单碱基插入突变 ,在 1972与 1973位核苷酸之间插入了一个腺嘌呤核苷酸 (A) ,使得密码子 6 2 4位以后的氨基酸发生了移码突变。在对照组未发现TSHR基因突变。结论　TSHR基因突变可能在自主性功能性甲状腺腺瘤的发病中起重要作用。