In-vitro studies of normal human thyroid cells: responses to thyrotrophin and dibutyryl cyclic AMP.

Follicular cells isolated from normal human thyroid tissue have been cultured for up to 140 h with bovine thyrotrophin (TSH) or dibutyryl cyclic AMP (DBcAMP). Both compounds induced marked reorganization of the cells into three-dimensional follicular structures, whilst non-supplemented cells assumed a monolayer form. Cultures treated initially with TSH or DBcAMP showed a greater iodide uptake capacity, in comparison with unsupplemented cultures, in which iodide uptake was markedly diminished after 24 h. The release of tri-iodothyronine (T3) and thyroxine (T4) into the medium was determined by radioimmunoassay. Both TSH- and DBcAMP-treated cells showed a significant increase in iodothyronine output compared with unsupplemented control cells. In contrast to the "classical" TSH-induced depression of the T4:T3 ratio in vivo, an increase in the ratio was observed for both TSH- and DBcAMP-supplemented cells in vitro. The ratio was also significantly greater after TSH than after DBcAMP, and possible implications of this findings are discussed.     

Modulation of thyrotrophin release from an intracellular pool by pre-exposure to thyrotrophin-releasing hormone and dibutyryl cyclic AMP

Amplification of desensitization of TSH response to thyrotrophin-releasing hormone (TRH) may be important mechanisms in the regulation of its secretion. We have investigated this possibility in vitro, using monolayer culture of rat anterior pituitary cells. Cells (1-1.5 X 10(5)/250 microliters per well) were cultured for 72 h, exposed to TRH or dibutyryl cyclic AMP (dbcAMP) for 6 or 8 h, washed, and then treated for 4 h with various doses of TRH, or with K+ (55 mmol/l) as a non-specific secretagogue. Pretreatment with TRH (20 nmol/l) for 8 h reduced subsequent TSH release: basal release fell to 64% of the control value (1.01 +/- 0.10 micrograms/l pretreated, 1.58 +/- 0.16 control) and release in response to TRH (100 nmol/l) to 69% of the control (2.7 +/- 0.19 micrograms/l vs 3.98 +/- 0.22); K+ response was reduced to 86% of the control (3.77 +/- 0.21 micrograms/l vs 4.39 +/- 0.20), significantly less than the other reductions. The extent of the parallel downward shift of the TRH dose-response curve was proportional to dose and duration of prior TRH exposure. There was no significant change in the dose of TRH required to cause half-maximal TSH release (ED50: pretreated 4.8, control 2.8 nmol TRH/l) suggesting depletion of an intracellular pool of TSH rather than 'desensitization'. After 6-h pretreatment with dbcAMP, subsequent TSH responses were augmented: basal release was 130% of the control, response to TRH (100 nmol/l) was 137% and to K+ it was 132% of the control, with a parallel upward shift of the TRH dose-response curve but no change in cellular TSH content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of forskolin on insulin release and cyclic AMP content in rat pancreatic islets

The effects of forskolin, a diterpene stimulator of adenylate cyclase, on cyclic AMP content and insulin release, have been studied in rat pancreatic islets. In the presence of 2.8 mM glucose, 0.3 to 30 microM forskolin gave concentration-dependent increases in cyclic AMP content to as much as seven-fold without causing any significant increase in insulin release. With 5.6 mM glucose, cyclic AMP contents were elevated by forskolin to the same extent as with 2.8 mM glucose but, under these conditions, insulin release was stimulated. 3 microM forskolin was a maximal concentration with respect to insulin release, but not for the elevation of cyclic AMP levels. Concentration-dependent increases in both insulin release and cyclic AMP content were detected over the range of 0.3 to 3 microM forskolin. The effect of forskolin to stimulate insulin release was rapid and was reversible within 15 minutes of removal of the drug. When tested with 16.7 mM glucose, forskolin potentiated both phases of stimulated release, with the greater effect upon the first phase.     

Forskolin and thyrotrophin stimulation of rat FRTL-5 thyroid cell growth: the role of cyclic AMP

The adenylate cyclase stimulator forskolin increases intracellular cyclic AMP (cAMP) in rat FRTL-5 cells within minutes and, after a lag phase of 20-24 h, an increase of cells in metaphase is seen. The dose-response relationships were similar in both systems, with significant increases in the number of metaphases observed at approximately 0.1 mumol/l and a doubling of cAMP levels at 1 mumol/l, whilst doses of 0.1 mmol/l and above proved cytotoxic. An involvement of intracellular cAMP as a positive intermediate in cell division was further suggested by the finding that a low dose of forskolin (0.1 mumol/l) potentiated TSH stimulation of mitosis. Isobutyl methyl xanthine (IBMX), a phosphodiesterase inhibitor, also acted as a mitogen and potentiated TSH action. Moreover, the simultaneous inclusion of low doses of IBMX and forskolin additionally potentiated TSH stimulation of mitosis. An analogue of cAMP, dibutyryl cAMP, also stimulated mitosis and acted over a restricted dose range, with maximal stimulation at 1 mmol/l. We conclude that cAMP may act as a positive signal for FRTL-5 thyroid cell proliferation.     

Adrenaline inhibition of insulin release: role of cyclic AMP.

Catecholamines inhibit adenylate cyclase in pancreatic B-cells, but the importance of the resulting fall in cAMP concentration for the decrease in insulin release remains controversial. Adrenaline caused a dose-dependent inhibition (EC50 = 5.7 nM) of insulin release by mouse islets incubated in a medium containing 15 mM glucose. Supplementation of the medium with 500 microM dibutyryl-cAMP or 1 microM forskolin potentiated the effect of glucose on release and attenuated the inhibition by 1 and 10 nM adrenaline; the EC50 value was increased 2-fold. The inhibitory action of 100 nM or 1 microM adrenaline was, however, not affected. This apparent change in adrenaline potency was not simply due to the larger rate of release since it was not observed when the effect of glucose was potentiated by cytochalasin-B. However, when the same rate of insulin release as that produced by 15 mM glucose alone was achieved by combining 10 mM glucose and 250 microM dibutyryl-cAMP, the inhibitory potency of adrenaline was unaffected. Intracellular microelectrodes were used to determine whether the changes in B-cell membrane potential brought about by adrenaline are mediated by a fall in cAMP levels. Addition of dibutyryl-cAMP or forskolin to a medium containing 10 or 15 mM glucose increased the Ca(2+)-dependent electrical activity triggered by the sugar. However, this did not prevent adrenaline from hyperpolarizing the membrane transiently and causing a steady-state decrease in the intensity of the electrical activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of thyroid-stimulating immunoglobulin-induced cyclic AMP accumulation in the rat thyroid cell strain FRTL-5: potentiation by forskolin and calibration against reference preparations of thyrotrophin

A clonal strain of rat thyroid cells (FRTL-5) has been used to investigate the biological activity of a Research Standard preparation of long-acting thyroid stimulator (LATS-B). Using the accumulation of intracellular cyclic AMP as a response parameter, significant stimulation was attained at a LATS-B dose of 0.75 mu./ml. The inter-bioassay coefficient of variation in response to a fixed dose of LATS-B (1.25 mu./ml) was 20.5%, as determined using eight sequential subcultures. Cells cultured directly from frozen stocks responded to both bovine TSH and LATS-B in a manner indistinguishable from cells subjected to regular subculturing. Cyclic AMP responses to incremental doses of LATS-B were potentiated after the inclusion of a low dose of forskolin (0.1 mumol/l). However, forskolin addition had no effect on the time-course of LATS-B-stimulated cyclic AMP accumulation, half-maximal responses being attained after 60 min in either the presence or absence of the diterpene. In the presence of 0.1 mumol forskolin/l, intracellular cyclic AMP responses to LATS-B were demonstrably parallel with those to human TSH (Second International Reference Preparation, 80/558), whilst parallel incremental cyclic AMP responses were also observed in respect of TSH and serial dilutions of a potent thyroid-stimulating immunoglobulin (TSIg) preparation, indicating that for this particular Graves' disease patient, TSIg bioactivity may be expressed in terms of a convenient and reproducible standard, as TSH microunit equivalents.     

Antagonistic influences of dibutyryl cyclic AMP and dibutyryl cyclic GMP on the beating rate of cultured mouse myocardial cells.

Cultured myocardial cells obtained from neonatal mouse ventricles beat spontaneously and rhythmically. Norepinephrine and dibutyryl cyclic AMP accelerated the rate of spontaneous beating. Dibutyryl cyclic GMP slightly decreased the rate of spontaneous beating. The acceleration of the beating rate of cultured myocardial cells by norepinephrine or dibutyryl cyclic AMP was counteracted by dibutyryl cyclic GMP. Preincubation of cultured myocardial cells with dibutyryl cyclic GMP prevented the positive chronotropic effect of dibutyryl cyclic AMP.     

Somatostatin partially impedes the stimulatory effects of thyrotrophin-releasing hormone and dibutyryl cyclic AMP on prolactin release: prolactin release through multiple routes.

Patterns of prolactin release were examined using stimulating and inhibiting agents. Primary cultured pituitary cells primed with oestrogens were used for perifusion experiments. TRH (100 nmol/l) increased the peak prolactin concentration to 360% of the basal concentration, while TRH, under inhibition by 1 nmol somatostatin/l, raised the peak prolactin concentration to 185% of the basal levels. When the somatostatin concentration was increased to 10, 100 and 1000 nmol/l, TRH still stimulated prolactin release to 128%, 121% and 140% respectively, indicating that concentrations of somatostatin of 10 nmol/l or higher did not further suppress the stimulatory effect of TRH. TRH (1 mumol/l) stimulated prolactin release under the influence of 0 (control), 1, 10, 100 and 1000 nmol dopamine/l (plus 0.1 mmol ascorbic acid/l) to 394, 394, 241, 73 and 68% of the basal concentration respectively, showing that the dopamine concentrations and peak prolactin concentrations induced by TRH have an inverse linear relationship in the range 1-100 nmol dopamine/l. The stimulatory effect of dibutyryl cyclic AMP (dbcAMP) on prolactin release was also tested. The relationship between dbcAMP and somatostatin was similar to that between TRH and somatostatin. When adenohypophyses of male rats were used for perifusion experiments, somatostatin (100 nmol/l) did not inhibit basal prolactin release from the fresh male pituitary in contrast with the primary cultured pituitary cells, but dopamine (1 mumol/l) effectively inhibited prolactin release. In conclusion, (1) oestrogen converts the somatostatin-insensitive route into a somatostatin-sensitive route for basal prolactin release, (2) TRH-induced prolactin release passes through both somatostatin-sensitive and -insensitive routes, (3) dopamine blocks both somatostatin-sensitive and -insensitive routes and (4) cAMP activates both somatostatin-sensitive and -insensitive routes.     

Hemodynamic effects of dibutyryl cyclic AMP in congestive heart failure

To evaluate the hemodynamic effects of dibutyryl cyclic AMP (DBcAMP) in congestive heart failure (CHF), right-sided cardiac catheterization was performed in 11 patients with CHF, and hemodynamic variables were investigated before and after infusion of various doses of DBcAMP at a rate of 0.025 to 0.2 mg/kg/min (mean 0.14 ± 0.077 [standard deviation]). DBcAMP reduced total systemic vascular resistance index from 3, 171 ± 1, 158 to 1,880 ± 554 dynes s cm^?5 · m² (mean ± standard deviation) and pulmonary arterial end-diastolic pressure from 23 ± 13 to 20 ± 11 mm Hg, and increased cardiac index from 2.24 ± 0.60 to 3.41 ± 1.02 liters/min/m². Mean arterial blood pressure decreased from 91 ± 14 to 84 ± 13 mm Hg, and heart rate increased from 91 ± 16 to 99 ± 13 beats/min. The increase in cardiac index was accompanied by a proportional decrease in total systemic vascular resistance index in all patients except 1. In 8 patients the decrease in pulmonary arterial end-diastolic pressure was accompanied by an increase or no change in the left ventricular stroke work index. In 6 patients, DBcAMP was given in incremental doses of 0.05, 0.1, and 0.2 mg/kg/min every 20 minutes, and 5 of 6 patients tolerated the full dose and showed dose-related hemodynamic changes for the incremental doses of DBcAMP. These data suggest that DBcAMP has powerful vasodilating effects on resistance vessels in patients with CHF; hence, it can be a useful vasodilating agent for treatment of CHF.     

Mechanisms of TSH release: studies with forskolin, phorbol ester and A23187

We have investigated the interactions of intracellular messenger systems which may regulate thyrotrophin (TSH) release in primary cultures of rat anterior pituitary cells. Calcium ionophore A23187 was used to raise intracellular free calcium, the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) to activate protein kinase C, and forskolin to activate adenylate cyclase. Each of these agents stimulated TSH secretion in a dose-dependent manner, but the effect of forskolin was delayed for at least 6 h. The combined effects of A23187 and TPA on TSH secretion were simply additive, but forskolin synergistically enhanced the effect of A23187 and 55 mM potassium, but not that of TPA. Co-incubation of cells with 1 microM A23187 caused parallel upward shift of the TPA dose-response curve, and the further addition of 10 microM forskolin led to further upward shift. These results suggest that calcium mobilization and adenylate cyclase activation may interact synergistically to modulate TSH secretion. Raised cellular cyclic AMP content may amplify further the TSH release stimulated by the combination of protein kinase C activation and raised intracellular free calcium.     

Potentiation of the inotropic effects of norepinephrine and dibutyryl cyclic AMP by theophylline

The positive inotropic response of the heart to catecholamines has been postulated to result from an increase in the intracellular level of cyclic AMP (adenosine 3′,5′-monophosphate), produced by activation of myocardial adenylate cyclase. According to this hypothesis, inhibition of phosphodiesterase, the enzyme which hydrolyzes cyclic AMP to inactive 5′-AMP, should enhance the cardiac effects of catecholamines. We therefore examined the effects of theophylline, an inhibitor of cardiac phosphodiesterase, on the isometric contractile response of cat papillary muscles to varying concentrations of norepinephrine (10⁻¹¹ to 10⁻⁵m), cyclic AMP (10⁻⁴ to 5 × 10⁻³m) and dibutyryl cyclic AMP (10⁻⁵ to 5 × 10⁻³m). The latter agent is a more lipid soluble derivative of cyclic AMP which is thought to reproduce the intracellular effects of the parent compound. Cyclic AMP produced no changes in isometric contractile function either in the presence or absence of theophylline. However, at a theophylline concentration (2.5 × 10⁻⁴m) which caused little increase in baseline contractile function (average increment in active tension less than 10%), a marked potentiation of the positive inotropic effects of norepinephrine and dibutyryl cyclic AMP was observed. The concentration of norepinephrine producing half-maximal increases in active tension and rate of tension development decreased from 10⁻⁷ to 10⁻⁸m while that of dibutyryl cyclic AMP decreased from 8 × 10⁻⁴ to 4 × 10⁻⁴m. Theophylline did not alter the positive inotropic effects of increasing extracellular calcium concentration (2.5 to 10 mm), an intervention which is not thought to directly influence intracellular cyclic AMP levels. Although indirect, these findings provide further evidence that the positive inotropic effects of catecholamines are mediated via the adenylate cyclase cyclic AMP system.