Prostacyclin stimulates the adenylate cyclase system of human thyroid tissue

Since Prostacyclin (PGI₂) is a major product of arachidonic acid metabolism in the human thyroid, we have studied the effects of PGI₂ on cAMP accumulation in human thyroid slices and cultured thyrocytes. In both systems, PGI₂ caused a dose- and time-dependent increase of cAMP accumulation with higher potency and efficacy than PGE₂. Two optically active isomers of 5,6-dihydro-PGI₂, i.e. stable synthetic analogs of PGI₂, had qualitatively similar effects to PGI₂. The relative potency ratio between the α- and β- isomer as well as their potency compared to PGI₂ were substantially similar to their potency in inhibiting human platelet aggregation. In thyroid slices, PGI₂ and its stable analogs had a greater than TSH in causing cAMP accumulation; however, in contrast to TSH, this effect was not associated with increased iodothyronine release except at maximal PGI₂ concentrations. TSH had no detectable effect on thyroidal PGI₂ synthesis and release. In cultured thyrocytes the effects of PGI₂ and its stable analogs were considerably less than those obtained with TSH and required higher concentrations. Such a discrepancy was not found in the case of PGE₂. These findings suggest the existence of a specific PGI₂-responsive adenylate cyclase system in human significance.     

Prostacyclin stimulation of adenylate cyclase activity in human thyroid membranes

Effect of prostacyclin (PGI2) on adenylate cyclase activity in human thyroid membranes was examined. PGI2 caused a dose- and time-dependent production of cyclic AMP (cAMP) with high potency. When GTP was added in concentrations up to 100 uM, the activation of adenylate cyclase by PGI2 was increased. In the assay medium containing 3 mM ATP, 10 uM GTP and nucleotide regenerating system, the replacement of Mg2+ by increasing concentrations of Mn2+ caused a progressive loss of PGI2 as well as TSH-stimulated adenylate cyclase activities, while high concentrations of Mg2+ (12 or 18 mM) slightly suppressed the activity stimulated by either PGI2 or TSH. Both agents had an additive effect on the stimulation of adenylate cyclase activity in the presence of either 6 mM Mg2+ or 6 mM Mn2+. Gamma-globulin fraction containing non-stimulatory TSH receptor antibody which was prepared from a patient with chronic thyroiditis, suppressed only TSH- but not PGI2-stimulation of the adenylate cyclase activity. These results suggest that PGI2 can stimulate the adenylate cyclase activity in human thyroid tissue, and that PGI2-stimulation may be mediated by the different system from TSH-dependent one.     

Forskolin stimulates adenylate cyclase and iodine metabolism in thyroid

Forskolin is a potent activator of the cyclic AMP-generating system in many tissues. In dog thyroid slices, the enhancement of cyclic AMP level was rapid, sustained in the presence of forskolin, but easily reversible after its withdrawal. Contrary to TSH, forskolin induced little apparent desensitization. Forskolin potentiated the effects of TSH, PGE1 and cholera toxin. However, the forskolin-induced cyclic AMP accumulation was still sensitive to inhibitors of dog thyroid adenylate cyclase such as iodide, norepinephrine and adenosine. As fluoride, but contrary to TSH and PGE1, forskolin stimulated adenylate cyclase in a medium where Mg2+ was replaced by Mn2+. This suggests that in thyroid, as in other tissues, forskolin acts beyond the receptor level but, as it potentiates hormone action and does not impair modulation by inhibitors, it may interact with the nucleotide-binding regulatory proteins. Forskolin mimicked the effect of TSH on iodide organification and secretion.     

Mono-ADP-ribosylation of Gs by an eukaryotic arginine-specific ADP-ribosyltransferase stimulates the adenylate cyclase system

An arginine-specific ADP-ribosyltransferase, named ADP-ribosyltransferase A, was partially purified from human platelets using polyarginine as an ADP-ribose acceptor. When human platelet membranes were incubated with the transferase A in the presence of NAD+, Gs, a stimulatory guanine nucleotide-binding protein of the adenylate cyclase was specifically mono-ADP-ribosylated. ADP-ribose transfer to Gs by this enzyme was suppressed when membranes were pre-ADP-ribosylated by cholera toxin. Incubation of membranes with the transferase A resulted in activation of the adenylate cyclase system. This stimulatory effect of the transferase A on the adenylate cyclase system was inhibited by the presence of polyarginine. These results indicate a role of ADP-ribosyltransferase A in regulation of the adenylate cyclase system via endogenous mono-ADP-ribosylation of Gs.     

Soluble adenylate cyclase from thyroid membranes

Adenylate cyclase from purified beef thyroid membranes has been solubilized by the use of Triton N-101 after preactivation with guanosine 5'-(beta, gamma-imido)-triphosphate. The soluble activity passed a 0.22- micron filter, was not sedimented at 100,000 X g for 2 h, and behaved like aldolase in sucrose density gradients and on Sepharose 6B. From comparison of the sedimentation in D2O and H2O the partial specific volume was found to be like that of globular proteins (0.75 +/- 0.006), hence little detergent appeared to be bound to the enzyme. The sedimentation coefficient was 7.4 +/- 0.15, the Stokes radius 45 A, and the molecular weight 159,000. Prestimulation by thyrotropin did not survive solubilization. The stimulation produced by guanosine 5'-(beta, gamma-imido)triphosphate persisted as did the more active state resulting from pretreatment with both this nucleotide plus thyrotropin. Thyrotropin did not stimulate the solubilized enzyme. The Km for ATP, thermal stability, and inhibition by Ca2+ were identical for the membrane-bound and soluble enzyme, while the pH optimum was increased 0.5 unit in the latter. Polyanions and phenothiazines inhibited both preparations equally, whereas only membranes responded to stimulation by polylysine and ribonuclease.     

Adenosine interactions with thyroid adenylate cyclase

Adenosine and certain adenosine analogues inhibit beef thyroid membrane adenylate cyclase. The inhibition has a rapid onset, is not directly on the catalytic or nucleotide regulatory sites, occurs with all activators tested (ITP, Gpp(NH)p, TSH, and F^?), and is seen also in mouse and human thyroid membranes. Addition of manganous ion, which activates adenylate cyclase, markedly enhances the inhibition by adenosine analogues. The order of potencies is: 2′,5′-dideoxyadenosine > 5′-deoxyadenosine > 2′-deoxy-3′-phosphoadenosine > 2′-deoxyadenosine > adenosine > adeninexyloside > adenine arabinoside. Purinemodified analogues are either inactive or stimulate slightly at high concentrations. This chemical specificity, the Mn²⁺ requirement, and the lack of reversal by theophylline, suggest that these membranes have little “R” site activity (stringent for the ribose moiety) and primarily contain a “P” site that has stringent purine requirement but permits changes in the ribose moiety. This site appears to be associated with the catalytic unit since it persists in solubilized adenylate cyclase.     

Inhibition of adenylate cyclase activity of human thyroid membranes by gangliosides

Gangliosides inhibit basal, thyrotropin-induced and fluoride-induced adenylate cyclase activity of human thyroid membranes in physiological conditions. In contrast neutral glycolipids, phospholipids and neuraminic acid containing oligosaccharides show no effect. The efficacy of inhibition is more dependent upon the position of the sialic acid residues than upon their absolute number. In general gangliosides with disialyl groups are more inhibitory than those with single sialyl moieties. The inhibitory effects of the individual gangliosides on the two modes of stimulation are parallel. This parallelism suggests that the inhibitory effect is located at the postreceptor level and that the gangliosides interact directly with the adenylate cyclase system. A possible role of thyroid membrane gangliosides as suppressive cofactors of adenylate cyclase is discussed in relation to recent findings of stimulating anti-ganglioside antibodies in Graves' disease.     

Calcium and calmodulin in the regulation of human thyroid adenylate cyclase activity.

TSH (thyrotropin)-stimulated human thyroid adenylate cyclase has a biphasic response to Ca2+, being activated by submicromolar Ca2+ (optimum 22nM), with inhibition at higher concentrations. Calmodulin antagonists caused an inhibition of TSH-stimulated adenylate cyclase in a dose-dependent manner. Inhibition of TSH-and TSIg-(thyroid-stimulating immunoglobulins)-stimulated activity was more marked than that of basal, NaF- or forskolin-stimulated activity. This inhibition was not due to a decreased binding of TSH to its receptor. Addition of pure calmodulin to particulate preparations of human non-toxic goitre which had not been calmodulin-depleted had no effect on adenylate cyclase activity. EGTA was ineffective in removing calmodulin from particulate preparations, but treatment with the tervalent metal ion La3+ resulted in a loss of up to 98% of calmodulin activity from these preparations. Addition of La3+ directly to the adenylate cyclase assay resulted in a partial inhibition of TSH- and NaF-stimulated activity, with 50% inhibition produced by 5.1 microM and 4.0 microM-La3+ respectively. Particulate preparations with La3+ showed a decrease of TSH- and NaF-stimulated adenylate cyclase activity (approx. 40-60%). In La3+-treated preparations there was a decrease in sensitivity of TSH-stimulated adenylate cyclase to Ca2+ over a wide range of Ca2+ concentrations, but most markedly in the region of the optimal stimulatory Ca2+ concentration. In particulate preparations from which endogenous calmodulin had been removed by La3+ treatment, the addition of pure calmodulin caused an increase (73 +/- 22%; mean +/- S.E.M., n = 8) in TSH-stimulated thyroid adenylate cyclase activity. This was seen in 8 out of 13 experiments.     

Roles of GTP and GDP in the regulation of the thyroid adenylate cyclase system

Effects of guanine nucleotides on the adenylate cyclase activity of thyroid plasma membranes were investigated by monitoring metabolism of the radiolabeled nucleotides by thin-layer chromatography (TLC). When ATP was used as substrate with a nucleotide-regeneratign system, TSH stimulated the adenylate cyclase activity in the absence of exogenous guanine nucleotide. Addition of GTP and GDP equally enhanced the TSH stimulation. Effects of GTP and GDP were indistinguishable in regard to their inhibitory effects on NaF-stimulated activities. The results from TLC suggested that GDP could be converted to GTP by a nucleotide-regenerating system. Even in the absence of nucleotide-regenerating system, addition of GDP to the adenylate cyclase assay mixture int he parallel decrease in ATP levels and formation of GTP indicating that thyroid plasma membrane preparatiosn possessed a transphosphorylating activity. When an ATP analog, App[NH]p, was used as substrate without a nucleotide-regenerating system, no conversion of GDP to GTP was observed. Under such conditions, TSH did not stimulate the adenylate cyclase activity unless exogenous GTP or Gpp[NH]p was added. GDP no longer supported TSH stimulation and caused a slight decrease in the activity. GDP was less inhibitory than Gpp(NH)p to the NaF-stimulated adenylate cyclase activity. These results suggest: (1) TSH stimulation of thyroid adenylate cyclase is absolutely dependent on the regulatory nucleotides. (2) In contrst to GTP, GDP cannot support the coupling of the receptor-TSH complex to the catalytic componenet of adenylate cyclase. (3) The nucleotide regulatory site is more inhibitory to the stimulation of the enzyme by NaF when occupied by Gpp[NH]p than GDP.     

Interaction of prostaglandin E2 with receptors and their effect on adenylate cyclase activity in human thyroid tissue

The investigations have been performed on thyroid tissue excised from patients suffering from euthyroid goiter and thyrotoxicosis. The study of thyroid thyrotoxic tissue has revealed the decrease in the number of free receptors to 3H-PGE2 caused by the enhanced production of receptor-endogenous PGE2 complexes whose level in thyrotoxic tissue was significantly higher than in euthyroid tissue. PGE2 and GppNHp had a pronounced stimulating effect on adenylate cyclaze activity both in euthyroid and thyrotoxic tissues, with enzyme sensitivity to stimulating effects considerably increased in the latter case.     

Parathyroid hormone stimulates adenylate cyclase in rat cerebral microvessels

We have studied the effect of parathyroid hormone (PTH) on adenylate cyclase of microvessels isolated from rat cerebral cortex. Native bovine (b) PTH-(1–84), the synthetic amino-terminal fragment bPTH-(1–34) and the synthetic analog [Nle⁸, Nle¹⁸, Tyr³⁴]-bPTH- (1–34) amide stimulated adenylate cyclase in a dose-dependent manner with apparent ED₅₀ values of 16 nM, 6.3 nM and 15 nM respectively. The stimulation by bPTH was greatly enhanced by guanosine triphosphate. The PTH antagonist, [Nle⁸, Nle¹⁸, Tyr³⁴]-bPTH-(3–34) amide inhibited the action of bPTH-(1–84) and bPTH-(1–34). In summary, PTH stimulated adenylate cyclase in rat cerebral microvessels in a very similar manner to its stimulation in the renal cortex.     

The adenylate cyclase system and calcitonin secretion from perfused dog thyroid lobes

The aim of the study was to assess the involvement of the adenylate cyclase system in calcitonin (CT) secretion from thyroidal C-cells. The cAMP analogues Br-cAMP (10(-6) and 10(-4) mol/l) and DB-cAMP (10(-4) mol/l) and the activators of adenylate cyclase cholera toxin (0.1 microgram/ml and 5 micrograms/ml) and forskolin (10(-7) mol/l and 10(-5) mol/l) were infused for 6 min periods in perfused dog thyroid lobes. CT was measured in thyroid effluent by radioimmunoassay. Br-cAMP and cholera toxin did not alter basal CT secretion. DB-cAMP had a minimal stimulatory effect and forskolin 10(-5) mol/l a moderate stimulatory effect. This was much less than the effect of increasing perfusate Ca++ from 1.5 to 2.0 mmol/l. 10(-4) mol/l Br-cAMP increased the response to Ca++ with approximately 50 per cent. These results suggest that the activity of the adenylate cyclase system of the C-cells by itself is of little importance for CT secretion, but that it may have a role as modulator of the response to Ca++.     

Corticotropin-releasing factor stimulates adenylate cyclase activity in the anterior pituitary gland

Ovine corticotropin-releasing factor (CRF) stimulates adenylate cyclase activity in rat anterior pituitary homogenate at an ED₅₀ value of 70 nM. GTP increases the stimulatory effect of CRF on [³²P] cyclic AMP formation in a rat adenohypophysial particulate fraction and in bovine anterior pituitary plasma membranes. The present data show that CRF stimulates adenylate cyclase activity in the anterior pituitary gland at least partly through a guanyl nucleotide-dependent mechanism.     

Effects of clofibrate on the human fat cell adenylate cyclase system.

The effects of chlofibrate on the adenylate cyclase system of human adipocytes were studied. Clofibrate reduced basal as well as hormone-NaF)stimulated adenylate cyclase activities to about the same extent (45% inhibition at 1 mg/ml clofibrate). The relative extent of hormonal stimulation was not altered by this compound. The inhibitory action of clofibrate was non-competitive with respect to the substrate ATP and cofactors (Mg2+-ions). Inhibition of enzyme activity was detectable after 2.5 min. Our results suggest that the antilipolytic activity of clofibrate is mediated via inhibition of the catalytic subunit of the fat cell adenylate cyclase.     

Multiple expressions of the activity of guanine nucleotide regulatory protein in human thyroid adenylate cyclase

Adenylate cyclase (ATP pyrophosphate-lyase, EC 4.6.1.1) in plasma membranes from human thyroid was highly responsive to thyrotropin. Pretreatment of thyroid plasma membranes with 5′-guanylylimidodiphosphate (Gpp(NH)p) in the presence of Mg²⁺ led to a temperature-dependent activation, which was seen neither in the absence of Mg²⁺ nor at 4 °C. By contrast, thyrotropin bound to its receptors regardless of the temperature and produced its maximal effect after 2 min of preincubation in the absence or presence of Mg²⁺. Furthermore, activation was seen after treatment with thyrotropin and Gpp(NH)p even carried out in the absence of Mg²⁺ or at 4 °C. However, the full activation by Gpp(NH)p required Mg²⁺, hormone, and elevated temperature. These observations suggest that there appears to be two types of nucleotide interaction responsible for the Gpp(NH)p activation in human thyroid membrane; one type seen in the absence of hormone may represent the system uncoupled from hormone receptor, while the fully coupled hormone-sensitive adenylate cyclase accounts for the second type of interaction which requires the presence of hormone.     

Properties of the adenylate cyclase system in the muscle tissue of the mollusk Anodonta cygnea

The adenylate cyclase system in the muscular tissue of Anodonta cygnea has been studied. A stimulating effect of serotonin, guanine nucleotides and sodium fluoride is found as well as a dependence of the catalytic activity of adenylate cyclase on magnesium and manganese ions. The mollusk enzyme and the vertebral one do not differ in principle in their functional properties. Only serotonin out of the tested biogenic amines (epinephrine, norepinephrine, dopamine, serotonin) stimulates the adenylate cyclase activity, which agrees with the fact that this neurohormone is a main neuromediator in mollusks. The analysis of the obtained results and data available in literature supports the idea about conservatism of the hormonal signal transfer in animals of different phylogenetic level.     

Lymphocyte adenylate cyclase and human aging

Adenylate cyclase activity was determined by enzymatic conversion of [32P]ATP to [32P]cAMP using peripheral lymphocytes freshly isolated from human subjects. The lymphocyte enzyme was stimulated by the potent beta-adrenergic catecholamine agonist isoproterenol and by the nonhydrolyzable GTP-analog Gpp[NH]p. The two activators had a synergistic effect, and agonist-dependent enzyme activity followed simple Michaelis-Menten kinetics with respect to isoproterenol in the presence but not in the absence of Gpp[NH]p. Cyclic AMP production by intact lymphocytes, determined by protein binding assay, also followed simple Michaelis-Menten kinetics with respect to isoproterenol. Kact of isoproterenol was the same in intact cells and the broken cell assay in the presence of Gpp[NH]p, suggesting the indispensable role the GTP-binding coupling factors play in the intact lymphocyte. In 31 human subjects between the age of 21 and 103, adenylate cyclase activity in the presence of isoproterenol, Gpp[NH]p, or isoproterenol in the presence of Gpp[NH]p decreased with the increasing age of the subject. The sensitivity of the enzyme to stimulation by isoproterenol, defined as the Kact and determined in the presence of Gpp[NH]p, was the same in lymphocytes from young (less than 45 years) or elderly (greater than 75 years) subjects. These results suggest a deficiency in the lymphocyte adenylate cyclase system distal to the beta-adrenergic catecholamine receptor could account for deterioration of cAMP-mediated components of the immune response which occur with age.     

Angiotensin stimulates adenylate cyclase activity via prostaglandins in the adrenal glomerulosa membrane

The effects of angiotensin II (A II) on adenylate cyclase activities in membranes of the zona glomerulosa (the capsular fraction) and the zona fasciculata (the decapsulated fraction) from rat adrenocortical glands were investigated. A time- and GTP-dependent stimulation by A II of adenylate cyclase activity was observed in the capsular fraction but not in the decapsulated fraction. The activation of adenylate cyclase by ACTH and A II was additive. Stimulation by A II was inhibited by an angiotensin antagonist, DD-3487 (DD). Moreover, the addition of a prostaglandin antagonist, a mixture of polyesters of polyphloretin phosphate (PPP) and an inhibitor of prostaglandin synthesis, indomethacin, was effective in inhibiting A II-induced stimulation of the capsular adenylate cyclase activity, suggesting that the activation of A II receptors located on the capsular membrane leads to the release of prostaglandins, which in turn stimulates the adenylate cyclase.