Evidence that organic iodine attenuates the adenosine 3',5'-monophosphate response to thyrotropin stimulation in thyroid tissue by an action at or near the adenylate cyclase catalytic unit

Studies were conducted to define more clearly the site in the thyroid adenylate cyclase complex at which iodine exerts its inhibitory effect on activation of this enzyme by TSH. Iodine- and TSH-induced desensitization were additive. Dissociation was observed between the rates of recovery from TSH- and iodine-induced desensitization. Cycloheximide (10(-4) M) prevented recovery from the inhibitory effect of iodine on thyroid adenylate cyclase activation. Preincubation of freshly isolated dog thyroid follicles in 10(-4) M iodide decreased the subsequent cAMP response to cholera toxin (0.5 micrograms/ml) stimulation. This effect of iodide was prevented by 3 mM methimazole. Thyroid adenylate cyclase regulatory protein (Ns) activity was assessed by the ability of detergent extracts of thyroid plasma membranes to reconstitute adenylate cyclase responsiveness to isoproterenol in N-deficient S49 cyc- plasma membranes. Thyroid Ns activities were similar in control and iodide-pretreated thyroid cells. The inhibitory effect of iodine on TSH activation of thyroid cAMP generation was additive to that of inhibition via the alpha 2- adrenergic pathway and also additive to inhibition by 2',5'-dideoxyadenosine (an adenosine P-site agonist). Preincubation of freshly dispersed dog thyroid cells in 10(-4) M NaI reduced the cAMP response to stimulation by 100 microM forskolin. These data provide evidence that in iodine-induced TSH desensitization in the thyroid; 1) TSH receptor function is normal, 2) the regulatory protein (Ns) in the adenylate cyclase stimulatory pathway is functionally unaltered, 3) iodine does not exert its effect via the regulatory protein (Ni) in the pathway that inhibits adenylate cyclase activation, 4) iodine does not act via the adenosine P-site inhibitory pathway, 5) the action of iodine is at or near the adenylate cyclase catalytic unit, and 6) new protein synthesis is necessary for recovery from iodine desensitization.     

Modulation of thyroglobulin release by dog thyroid slices

Nonbutanol-extractable 131I (NBE131I) release by dog thyroid slices in vitro has been shown previously to be primarily thyroglobulin (Tg); it is stimulated by TSH. NBE131I (Tg) release has therefore been considered as an in vitro model of thyroglobulin secretion and was further characterized in this work. TSH-stimulated NBE131I (Tg) release, like TSH-stimulated BE131I (T4, T3 and iodide) release was reproduced by forskolin, an activator of adenylate cyclase. TSH-, (Bu)2cAMP- and forskolin-stimulated NBE131I (Tg) release was inhibited by 10(-5) M carbamylcholine, an effect relieved by 10(-5) M atropine, but not by 10(-4) M 1-methyl-3-isobutylxanthine. NBE131I (Tg) release was observed in the presence of 2 mM methimazole and 2 mM perchlorate. Cooling the slices to 20 degrees C or addition of 10(-5) M monensin completely blocked the formation of apical pseudopods and BE131I release but not NBE131I (Tg) release. Inhibition by 500 microM chloroquine of intralysosomal Tg hydrolysis and BE131I release did not enhance NBE131I (Tg) release. Cytochalasin B induced a concentration-dependent increase in basal and TSH-stimulated NBE131I (Tg) release at concentrations which depressed TSH-stimulated BE131I release. Removal of Ca2+ from the medium and slices by 10(-3) M or 10(-4) M EGTA increased NBE131I (Tg) release. In conclusion, in dog thyroid slices, TSH-stimulated NBE131I (Tg) release was mediated by cAMP and inhibited by 10(-5) M carbamylcholine at a step beyond cAMP. It was not neosynthesized Tg. It did not seem to require the formation of apical pseudopods or to result from the escape from lysosomes of undegraded thyroglobulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of proteolytic enzymes and protease inhibitors on bovine thyroid adenylate cyclase activity

Trypsin, chymotrypsin, and papain stimulate basal adenylate cyclase activity in bovine thyroid plasma membranes in a dose-related, albeit biphasic, fashion. Each of the proteases enhanced TSH-stimulated adenylate cyclase activity over basal activity. The proteases also enhanced GTP-, guanosine 5'-(beta, gamma-imidotriphosphate)-, prostaglandin E1-, and cholera toxin-stimulated adenylate cyclase to varying degrees. Fluoride-stimulated activity was enhanced by chymotrypsin and papain, but not by trypsin. When Mn++ was substituted for Mg++ in the adenylate cyclase assay, no stimulation by the proteases were observed. To see if endogenous membrane proteases are required for optimal thyroid adenylate cyclase response to TSH and other stimulators, studies were performed using the protease inhibitors tosylamide 2-phenylethyl-chloromethyl ketone (TPCK) and p-tosyl-L-arginine methyl ester (TAME), inhibitors of chymotrypsin and trypsin, respectively. TPCK (0.15 mM) had no effect on basal adenylate cyclase activity, but did inhibit TSH-, trypsin-, and chymotrypsin-stimulated activities by approximately 90%. Guanosine 5'-(beta, gamma-imido) triphosphate- as well as cholera toxin-stimulated activities were inhibited by approximately 50%, whereas prostaglandin E1- and fluoride-stimulated activities were inhibited by approximately 25%. TAME (6 mM) produced similar results, except that no effect on fluoride activity was seen, while basal activity was inhibited by approximately 20%. Thus, various serine proteases augment both basal and hormone-stimulated adenylate cyclase in bovine thyroid. Since both trypsin- and chymotrypsin-stimulated as well as TSH-induced enzyme activities were inhibited by TPCK and TAME, it would appear that augmentation of thyroid adenylate cyclase activity may, in part, result from stimulation of endogenous proteases.     

Forskolin stimulation of adenylate cyclase in human thyroid membranes

Forskolin stimulates adenylate cyclase in human thyroid membranes approximately 7-fold with half-maximal stimulation occurring at 5-10 microM. Guanine nucleotides are not required for stimulation of the enzyme by forskolin. Forskolin-stimulation is additive or greater than additive with that of TSH or Gpp(NH)p- (above 1 microM). Different from TSH- or Gpp(NH)p-stimulation of adenylate cyclase, uncoupling of the guanine nucleotide-binding regulatory component by increasing concentrations of MnCl2 did not result in uncoupling of forskolin stimulation. The finding indicates that forskolin may mainly act on the catalytic component of adenylate cyclase. From the present study, it is suggested that the diterpene forskolin stimulates adenylate cyclase in human thyroid membranes by a novel mechanism that differs from TSH- or Gpp(NH)p-stimulation, and that the diterpene may be a useful tool to investigate the metabolism of thyroid and its regulation in normal and pathological situations.     

Basal and stimulated adenylate cyclase activity in the gill epithelium of the rainbow trout

Basal and stimulated adenylate cyclase specific activity was characterized in gill plasma membrane of freshwater-adapted trout by measuring the conversion of [alpha-32P]ATP into [alpha-32P]cyclic AMP. Both basal and isoproterenol- or sodium fluoride-stimulated enzyme activities were linear with time and protein concentration. The optimum activities were obtained using a pH buffer of 7.5 and a temperature of 20 degrees. The Km for ATP was 0.5 mM in the presence or absence of the stimulators. The presence of 10(-5) M guanosine-5'-triphosphate and 4 X 10(-3) M MgCl2 (2.41 X 10(-3) M free Mg2+) was required to optimize not only the basal activity but also the stimulation ratio (test/control) produced by these agents. On the contrary, Ca2+ was inhibitory. IC50 for CaCl2 was 5 X 10(-4) M (10(-7) M free Ca2+) in the presence or absence of the stimulators. Under these conditions, the basal adenylate cyclase specific activity was 400-450 pmol/mg protein/10 min. A maximal stimulation was produced by isoproterenol or PGE1 10(-5) M (50% increase over basal activity) or by glucagon 5.7 X 10(-10) M (30%). In addition, this enzyme displayed high sensitivity to sodium fluoride which induced a particularly large maximal effect (370%) at a concentration of 10(-2) M.     

Skin-derived fibroblasts respond to human parathyroid hormone-like adenylate cyclase-stimulating proteins

Human tumors and keratinocyte-conditioned medium contain PTH-like adenylate cyclase-stimulating proteins. Human dermal fibroblasts have receptors that recognize PTH and a factor associated with humoral hypercalcemia of malignancy in rats. We examined 10 human dermal fibroblast lines for an adenylate cyclase response to PTH. Six of 10 lines tested displayed a definite response (2.4- to 3.8-fold over basal) to 10(-6) M bovine PTH-(1-34). This response was inhibited by the PTH analog and antagonist Nle8,18,Tyr34-bPTH-(3-34). We also examined whether human dermal fibroblasts are capable of responding to either a human PTH-like tumor-derived factor or the PTH-like factor contained in human keratinocyte-conditioned medium. Both human humoral hypercalcemia of malignancy-associated tumor extract (2.5 X 10(-10) M) and keratinocyte-conditioned medium (8 X 10(-10) M) stimulated human dermal fibroblast adenylate cyclase. These concentrations are markedly lower than those required for PTH-induced adenylate cyclase stimulation. This activity was also inhibited by the PTH analog. The high prevalence of PTH-responsive adenylate cyclase in dermal fibroblast lines and the apparent potency of tumor-derived and keratinocyte-derived PTH-like factors in dermal fibroblasts suggest that these factors may play a role in normal dermal physiology.     

Thyrotrophin-responsive adenylate cyclase activity in thyroid toxic adenoma.

The adenylate cyclase system was studied in hyperfunctioning autonomous nodules in comparison with normal thyroid tissue. The basal, TSH- and NaF-stimulated adenylate cyclase activities were tested in purified plasma membrane preparations. Basal enzyme activity in membranes from hyperfunctioning nodules was variable and the response to TSH was either normal, low or absent. The present study demonstrates that an intact adenylate cyclase activity, hyporesponsive to TSH, may exist in the cell membrane of the adenoma.     

Adenylate cyclase system responsive to thyroid stimulating hormone (TSH) of porcine thyroid cells in primary monolayer cultures. Potential effect of forskolin on TSH-mediated adenylate cyclase stimulation

The TSH-responsive adenylate cyclase system was studied using porcine thyroid cells in a primary monolayer culture. Isolated porcine thyroid cells treated with collagenase were inoculated into 96 wells at the density of 5 X 10(4) viable cells/0.25 ml Ham F-12 containing 10% fetal bovine serum and cultured for 4 days in a humidified atmosphere with 5% CO2. Adenylate cyclase activities in the cells treated or non-treated with protein synthesis inhibitor were assayed in Hanks/20 mM Hepes buffer (pH 7.4) containing 1% BSA, 1 mM IBMX and various stimulants at 37 degrees C for 30 or 60 min. The reaction was stopped by adding ice-cold TCA, and cAMP content in the extract was measured by radioimmunoassay after treatment with water-saturated ether. The cultured thyroid cells had an adenylate cyclase system responsive to TSH, cholera toxin and forskolin. TSH (50 mU/ml) stimulated the activity about eight fold over the basal activity. Cholera toxin (1 microgram/ml) and forskolin (100 microM), however, were much stronger activators of the adenylate cyclase system. In the cells pretreated with cyclo-heximide (5 micrograms/ml) up to 24 hours, cAMP formation by TSH was potentiated 200 approximately 170% compared to that in non-treated cells, suggesting a suppression of an inhibitory mechanism dependent upon new protein synthesis. In contrast, forskolin (100 microM)-stimulation was greatly reduced to 30% of the control after 24-hour treatment. Cholera toxin (1 microgram/ml)-stimulation was significantly lessened or slightly reduced by the treatment. Although the ability of forskolin to act synergistically with TSH or cholera toxin was observed in non-treated cells, it was clearly unaffected and demonstrated in the cells treated with protein synthesis inhibitor. The mechanism(s) and site(s) of forskolin action still remain unclear. However, these observations are compatible with a two-site model of forskolin action. The direct activating site of forskolin appears to reside in a protein which is closely associated with the catalytic unit of adenylate cyclase system and has a relatively shorter half-life than other components of the system. The potential action of forskolin may reside in a more stable complex of an activated stimulatory guanine nucleotide binding component and catalytic unit of the adenylate cyclase system. Based on these results, it is likely that the primary monolayer culture of porcine thyroid cells is a good model to investigate the adenylate cyclase system in the thyroid, and that forskolin may potentiate the TSH-mediated stimulation of adenylate cyclase.     

Dual activation by thyrotropin of the phospholipase C and cyclic AMP cascades in human thyroid

In human thyroid slices prelabeled with myo-[2-3H]inositol, thyrotropin (TSH, 3-30 mU/ml) stimulated IP3, IP2 and IP1 generation over a prolonged time course. The cAMP response was much more sensitive to TSH, peaking between 1 and 5 mU/ml. Forskolin (10(-5) M) and isoproterenol had no effect on basal IP levels, while carbamylcholine (10(-5) M, 10(-4) M) also increased IP accumulation. These data suggest that in the human thyroid, TSH activates a phospholipase C generating IP3 and diacylglycerol independently of the well-known adenylate cyclase stimulation. They validate in the human model a dual mode of action of the hormone previously proposed on the basis of indirect observations.     

TSH-responsive adenylate cyclase activity in thyroid tissue from patients with Graves' disease

Basal adenylate cyclase activity of thyroid plasma membranes obtained from six patients with Graves' disease was slightly but not significantly lower than normal (83.3 +/- 13.9 pmol cAMP/10 min/mg of protein versus 120.9 +/- 19.5 pmol cAMP/10 min/mg of protein). In five of these patients the adenylate cyclase activity was stimulated by bovine TSH with an apparent Km value similar to that of normal thyroid (3.1 +/- 0.5 X 10-9 M versus 3.4 +/- 0.6 X 10-9 M). The response to prostaglandin E2 was also normal. In the sixth patient adenylate cyclase activity was stimulated by prostaglandin E2 but not by bovine TSH. The distribution of basal adenylate cyclase activity in various gradient layers was studied in two TSH-responsive patients. A relative increase of this activity was found in the denser layer when compared to normal thyroid tissue. This could be the expression of an altered ratio between the protein and lipid components of the plasma membranes in patients with Graves' disease.     

Intralysosomal hydrolysis of thyroglobulin. II. Different fates of poorly and fully iodinated Tg and specific activation by TSH of the degradation of fully iodinated Tg

Even though adrenergic nerve terminals between and around thyroid follicles and catecholamine stimulation of thyroid adenylate cyclase have been reported, there is no uniform concept on catecholamine interaction with thyrotrophin (TSH) receptors. Therefore, the effect of catecholamines on TSH-stimulated cyclic AMP (cAMP) accumulation in human follicular thyroid cells has been investigated, to thus eliminating the extrathyroidal actions of catecholamines. Epinephrine, norepinephrine and isoproterenol appeared to be rapid and potent stimulators of intracellular cAMP accumulation, the half maximum increase doses being 4 X 10(-7)M, 1 X 10(-5)M and 5 X 10(-7)M, respectively. While propranolol (1 X 10(-5)M) prevented the stimulatory effect of catecholamines and failed to inhibit the effect of bovine TSH, phentolamine (1 X 10(-5)M) enhanced the potency of norepinephrine and bovine TSH, leaving that of epinephrine unchanged. The effects of epinephrine (2 X 10(-8)M) and isoproterenol (2 X 10(-8)M) were additive to that of bovine TSH (0.5 mU/ml), but the effect of simultaneous stimulation with norepinephrine (5 X 10(-7)M) and bovine TSH (0.5 mU/ml) was lower than expected. Prenalterol, a selective beta 1-agonist, did not stimulate cAMP accumulation, while terbutaline, a selective beta 2-agonist, exerted a potent stimulation. Metoprolol, a selective beta 1-adrenergic blocker, did not affect the response of thyroid follicular cells to isoproterenol. These results demonstrate the existence of beta-adrenergic receptors in human thyroid follicular cells, mainly of the type beta 2, apparently not correlated with TSH receptor. The existence of alpha-adrenergic receptors which counter-regulate TSH functional responses in human thyroid follicular cells is suggested.     

Inhibition of cyclic AMP formation by iodide in suspension cultures of porcine thyroid follicle cells

In the present study porcine thyroid cells in suspension cultures were employed to investigate the suppressive effect of iodide on adenylate cyclase under basal conditions and following incubation with TSH, PGE1, cholera toxin and forskolin. Within 30 min of incubation with iodide (half-maximal effect 10(-5) M), inhibition was established and remained unchanged up to 40 h of culture. The inhibitory action was abolished by methimazole. TSH, PGE1, cholera toxin and forskolin stimulated cAMP accumulation 10-, 3-, 24- and 22-fold, respectively. Iodide pretreatment reduced basal cAMP levels and also made the cells less sensitive to stimulation by the various agents. High concentrations of TSH or PGE1 could not overcome the suppressive influence of iodide, whereas with high concentrations of cholera toxin and forskolin the reduction in cAMP levels in iodide-treated cultures was less pronounced. Membranes isolated from iodide-treated cultures produced significantly lower amounts of cAMP compared to control membranes. Furthermore, iodide did not inhibit basal or forskolin-stimulated cAMP production in human fibroblasts. The results demonstrate that iodide via an iodination-dependent mechanism influences cAMP generation in thyroid cells. It is suggested that the inhibitory activity, which has a long half-life, involves stable modification of the membrane-localized catalytic unit of adenylate cyclase such that its activation by the regulatory unit is rendered less efficient.     

Role of cellular Ca++ in phosphorylation of 21 K and 19 K polypeptides in cultured thyroid cells: effects of phorbol ester, trifluoperazine, and 8-diethylamino-octyl-3,4,5-trimethoxybenzoate hydrochloride

Cultured dog thyroid cells contain 21 and 19 kilodalton (K) phosphoproteins which by several criteria have been identified as light chains of myosin (MLC). TSH causes a reduction in the phosphorylation state of the 21 K-19 K proteins, at least in part through activating adenylate cyclase and increasing cAMP levels. We now report that 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also decreases the 21 K-19 K protein phosphorylation state, but in contrast to that due to TSH, the TPA-induced decrease is not associated with elevated cAMP levels. The effect of TPA was not additive to that of TSH. Because Ca++ is a major factor regulating MLC kinase and TPA-stimulated protein kinase C in other systems, the role of Ca++ in the phosphorylation of the 21 and 19 K polypeptides in dog thyroid was examined. In intact cells, both (8-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) (1 X 10(-4) M) and trifluoperazine (TFP) (4 X 10(-5) M) increase basal 21 K-19 K protein phosphorylation and inhibit the decrease in phosphorylation caused by TSH and TPA without affecting cAMP levels. Ionophore A23187 (5 X 10(-6) M) counteracts TMB-8- and TFP-stimulated phosphorylation as well as TMB-8 and TFP inhibition of TSH- and TPA-reduced 21 K-19 K phosphorylation. Incubation of 32PO4-labeled dog thyroid cells in the absence of extracellular Ca++ or with verapamil does not significantly affect basally phosphorylated 21 K-19 K proteins or the decreased 21 K-19 K phosphorylation state caused by TSH. These results strongly suggest that the phosphorylation state of the 21 and 19 K proteins is affected more significantly by intracellular Ca++ pools than by extracellular Ca++, and implicate a kinase(s) other than Ca++-calmodulin-dependent MLC kinase in the phosphorylation of MLC in the dog thyroid.     

Adenosine 3',5'-monophosphate-dependent protein kinase activity in soluble thyrotropin receptor complex.

cAMP-dependent protein kinase activity was present in a soluble TSH receptor fraction. The Km of this enzyme was 2.2 X 10(-6) M for casein substrate in the absence or presence of 10(-5) M cAMP. A [3H]cAMP-binding protein was also found in this fraction. The Ka for [3H]cAMP-binding was 0.11 X 10(6) M-1, with a total binding capacity of 3 nmol/mg protein. After fractionation using a continuous sucrose density gradient, one of the several [125I]iodobovine TSH-binding peaks corresponded to a [3H]cAMP-binding peak. After fractionation on a sucrose density gradient containing 0.4 M NaCl at pH 6.5, a major peak of protein kinase activity was shown. This protein kinase activity was stimulated by adding 10(-5) M cAMP. A peak of [3H]cAMP-binding activity corresponded to the same peak. Protein kinase activity in the receptor fraction was stimulated by adding 6 mg/ml bovine TSH. The soluble TSH receptor fraction also has an adenylate cyclase activity stimulated by TSH. These results suggest that some TSH receptors released from thyroid plasma membranes have associated adenylate cyclase activity and cAMP-dependent protein kinase activity. The receptor, cyclase, and kinase activities may exist in a functional primary receptor unit which is spontaneously released from plasma membranes.     

Inhibitory action of adenosine on histamine- and dopamine-stimulated cardiac contractility and adenylate cyclase in guinea pigs

Infusion of adenosine into the coronary arteries of isolated guinea pig hearts produced a dose-dependent inhibition of cardiac contractile force development elicited by bolus injections of histamine (7.5 X 10(-9) mol) or dopamine (1.5 X 10(-8) mol). Threshold concentration of adenosine was 10(-7) M and maximal inhibition (90%) was obtained at 3 X 10(-5) M. Adenosine in the effective concentration range did not alter Ca2+-induced increases in contractile force. The rise in tissue levels of cAMP induced by equieffective doses of histamine (7.5 X 10(-9) mol) and dopamine (1.5 X 10(-8) mol) was inhibited by adenosine (3.5 X 10(-5) M) by about 60%. In a particular membrane preparation of guinea pig ventricles the adenylate cyclase activity stimulated by the histamine (10(-5) M) and dopamine (10(-4) M) was inhibited in a dose-dependent manner by adenosine. This effect could be reversed by theophylline (5 X 10(-5) M) in a competitive manner. The hormone-insensitive adenylate cyclase of a Lubrol-PX solubilized membrane preparation stimulated by NaF or 5'-guanylylimido-diphosphate (GppNHp) was also inhibited by adenosine (40% and 90% inhibition at 10(-5) M and 10(-4) M, respectively). Adenosine did not influence the Km value of the adenylate cyclase for ATP, but markedly lowered Vmax of the enzyme. From additional studies with purine-substituted (N6-methyl-adenosine, N6-phenylisopropyl-adenosine) and ribose-substituted (2'-deoxy-adenosine and arabino-furanosyl-adenine) adenosine analogues, we conclude that adenosine may inhibit the inotropic responses to hormones as well as the adenylate cyclase activity by specifically interacting with at least two different sites associated with the adenylate cyclase.     

Actions of growth hormone-releasing factor and somatostatin on adenylate cyclase and growth hormone release in rat anterior pituitary

The interaction of growth hormone-releasing factor (GRF) and somatostatin (SRIF) on adenylate cyclase activity and growth hormone release was investigated in pituitary homogenates and 2-day cultured rat anterior pituitary cells. GRF stimulated growth hormone release by about 3-fold (ED50 1.6 X 10(-12) M) and caused a rapid 15-fold increase in cyclic AMP production (ED50 6.0 X 10(-12) M). The increase in cyclic AMP was due to direct stimulation of adenylate cyclase by GRF, which caused a 4-fold increase in the activity of the enzyme measured in anterior pituitary homogenates. GRF-induced cyclic AMP formation and GRF-stimulated adenylate cyclase activity were maximally inhibited to the extent of about 50% by 10(-8) M somatostatin. In contrast, GRF-stimulated growth hormone release was completely inhibited by somatostatin (ID50 3.2 X 10(-11) M), suggesting a second site of action of somatostatin. These studies demonstrate that GRF stimulates growth hormone release via activation of adenylate cyclase and a rise in intracellular cyclic AMP. In addition, these findings indicate that the inhibitory action of somatostatin on growth hormone release is exerted at two levels, one at the level of adenylate cyclase affecting the production of cyclic AMP, and the other beyond the formation of the nucleotide, at a site which modulates the release of growth hormone from the cell.     

Growth hormone-releasing factor-sensitive adenylate cyclase system of purified somatotrophs: effects of guanine nucleotides, somatostatin, calcium, and magnesium

The purpose of this study was to characterize the adenylate cyclase system in a purified population of normal somatotrophs derived from rat pituitary and to determine the responses of this system to GRF, somatostatin, guanine nucleotides, and cations. Additionally, experiments were performed to evaluate the interrelationships among changes in adenylate cyclase activity, cellular cAMP levels, and GH release induced by GRF and somatostatin. The results obtained using homogenates and membrane preparations from somatotrophs included the following. 1) GRF caused guanine nucleotide-dependent concentration-related (Ka, approximately 10(-8) M) stimulation of adenylate cyclase activity. 2) Guanine nucleotides were effective in stimulating cyclase in the absence of GRF; the concentration of guanine nucleotide required for half-maximal stimulation was decreased more than 10-fold in the presence of GRF. 3) Adenylate cyclase activity increased with increasing concentrations of free Mg2+ (0.25-20 mM); activation by GRF and guanine nucleotide resulted in an approximately 7-fold increase in the enzyme's affinity for free Mg2+. 4) Somatostatin, up to 10(-6) M, did not alter basal or GRF-stimulated adenylate cyclase activity. 5) Ca2+ (0.5-11.9 microM) produced concentration-dependent inhibition of basal (up to 28%) and GRF-stimulated (up to 47%) cyclase activities; the inhibitory effect of Ca2+ was accompanied by a decrement (2- to 3-fold) in the apparent affinities of the enzyme for both GRF and guanine nucleotide. In intact somatotrophs, GRF produced concentration-dependent stimulation of GH release (Ka, approximately 6 x 10(-11) M), preceded by a marked elevation of cAMP levels. While somatostatin blocked GRF-induced GH release, the augmented cAMP levels were only slightly reduced.(ABSTRACT TRUNCATED AT 400 WORDS)     

Desensitization of the thyroid cyclic AMP response to thyroid stimulating immunoglobulin: comparison with TSH

Studies were conducted to examine the characteristics of thyroid cell cAMP stimulation by thyroid stimulating immunoglobulins (TSI) and to compare the cAMP response to TSI and TSH in desensitized human thyroid cells. In terms of cAMP production, preexposure (eight hours) of the cells to TSI induced a desensitization very similar to TSH-induced desensitization: both TSH- and TSI-desensitized cells showed a normal response to cholera toxin and forskolin stimulation; TSH and TSI desensitization was interchangeable in that desensitization by either stimulator affected the action of the other; the time of recovery from either TSH and TSH desensitization was identical; the cycloheximide (10(-4) mol/L) prevented both TSI- and TSH-induced desensitization; preexposure of the cells to iodine, which affects mainly the adenylate cyclase catalytic unit, or to epinephrine, which activate the inhibitory regulatory protein Ni by the alpha 2-adrenergic stimulation, induced a similar inhibition of the subsequent stimulation by both TSH or TSI. The remarkable similarities between TSH and TSI in stimulating and desensitizing thyroid cells strongly support the concept that TSI activates thyroid adenylate cyclase by interacting with the TSH receptor and not through an allosteric mechanism.     

Thyrotropin receptor-adenylate cyclase system in plasma membranes from normal and diseased human thyroid glands.

Thyrotropin binding characteristics and adenylate cyclase (AC) activity of thyroid plasma membranes were studied in 52 tissues from normal and diseased human thyroids. Data from normal glands, Graves' goiters, non toxic multinodular goiters and nodular and perinodular tissue of toxic nodular goiters show the same basal, TSH- and NaF- stimulated adenylate cyclase activities (no. = 45; 34.1 +/- 3.2 (m +/- SE), 378 +/- 43, 298 +/- 48 pmol cAMP x min-1 x mg membrane protein-1), the same stimulability of AC by TSH (11.3 +/- 1.4--fold over basal level) and by NaF (8.1 +/- 1.8-fold), the same apparent TSH binding equilibrium constants (5.6 +/- 0.7 and 406 +/- 57 nM) and the same TSH binding site concentrations (2.2 +/- 0.4, 27.8 +/- 5.9 pmol x mg membrane protein-1). Alterations of the TSH receptor and of the AC were detected in membranes from tumoral and metastatic lymph node tissues from thyroid papillary carcinoma and in the thyroid tissue from post-radioiodide therapy thyroiditis. These observations suggest that: (i) hyperthyroidism in Graves' disease or toxic nodular goiter does not result in and is not a consequence of an alteration in the TSH receptor-adenylate cyclase system; (ii) there is no evidence supporting a relationship between the studied membrane properties and clinical or histological status; (iii) membrane abnormalities detected in thyroid carcinoma vary widely; (iv) studies of these membrane alterations might be of interest in the therapeutic management of thyroid carcinoma and may lead to a better understanding of the receptor-adenylate system.     

Decrease in cAMP-dependent protein kinase activity in suspension cultures of porcine thyroid cells exposed to TSH or forskolin

Suspension cultures of porcine thyroid cells were used to study the action of TSH and forskolin (Fk) on cAMP-dependent (PKa) and Ca2+-phospholipid-dependent (PKc) protein kinase--enzymes which represent the key step in the transduction of extracellular signals. The PKa activity in cells cultured for 2 days in the presence of TSH was decreased to about 50% of control level with a TSH dose of 0.1 mU/ml. This decrease is dose dependent; only traces of PKa activity remained at very high doses of TSH (50 mU/ml). Similar results were obtained with Fk (10(-5) M), the adenylate cyclase activator. It decreased the PKa activity to the level obtained with 0.1-1.0 mU/ml TSH. The loss of the PKa activity was parallel in cytosol and particulate fractions, suggesting that there is no translocation of enzymes under the action of either TSH or Fk. Neither TSH nor Fk had any effect on PKc, which became the predominant activity in cells exposed to either of the regulators. The cAMP-dependent phosphorylation of endogenous proteins was lower in TSH- or Fk-treated cells than in controls, and was dependent, like the PKa activity, on the dose of TSH. Polyacrylamide gel electrophoresis (PAGE) revealed the specific substrates of PKa in cultured thyroid cells. Proteins of 28, 30 and 33 kDa were regularly found, while 58 kDa protein was not present in all experiments. PAGE patterns showed that the decrease in endogenous phosphorylation in TSH- and Fk-treated cells was due to decreased labelling of PKa-specific substrates. The observed down-regulation of PKa activity could have an influence on the expression of thyroid cell differentiation.