Association of a 68,000-dalton protein with adrenocorticotropin-sensitive adenylate cyclase activity in Y1 adrenocortical tumor cells

This report explores the biochemical basis for clonal variation in adrenocorticotropin (ACTH)-sensitive adenylate cyclase activity in the Y1 mouse adrenocortical tumor cell line. We demonstrate that the level of a specific protein, designated p68, is significantly correlated with the ability of adrenocorticotropin to stimulate adenylate cyclase activity among Y1 subclones (p = 0.004; r = 0.65). p68 was characterized by its molecular weight in sodium dodecyl sulfate polyacrylamide gels (Mr = 68,000) and by its isoelectric point as determined by two-dimensional gel electrophoresis (pI = 7.2). On two-dimensional gels, the protein migrated as a major spot with satellite spots 0.1 pH unit on either side. Homogenates and plasma membrane fractions from clones highly responsive to ACTH had large amounts of p68. In homogenates from highly responsive clones p68 represented 10 to 12% of the total protein. Homogenates and plasma membrane fractions from clones insensitive to ACTH were deficient in p68. In homogenates from the insensitive clones Y6 and OS3, p68 represented less than or equal 0.8% of the total protein. A somatic cell hybrid, formed by fusion of these two ACTH-insensitive clones recovered ACTH-sensitive adenylate cyclase activity and concomitantly expressed appreciable levels of p68. It is suggested that p68 may regulate the transfer of information from the occupied ACTH receptor ot the catalytic subunit of adenylate cyclase.     

Heterologous desensitization of the human dopamine D1 receptor in Y1 adrenal cells and in a desensitization-resistant Y1 mutant.

In previous studies, mutant clones (designated Y1DR) were isolated that resisted ACTH-induced homologous desensitization of adenylyl cyclase. The Y1DR mutation also conferred resistance to the homologous desensitization induced by agonist stimulation of transfected beta 2-adrenergic receptors. These observations suggested that ACTH and beta 2-adrenergic agonists homologously desensitized adenylyl cyclase in Y1 cells by a common mechanism. In the present study, parental Y1 cells (Y1DS) and the Y1DR mutant were transfected with the gene encoding the human dopamine D1 receptor and examined for regulation of adenylyl cyclase by dopaminergic agonists. Transformants were isolated from both cell lines and shown to respond to dopamine agonists with increases in adenylyl cyclase activity. Treatment of the Y1DS transformants with ACTH promoted a rapid, homologous desensitization of adenylyl cyclase and had little effect on the responses to dopamine or NaF; treatment of Y1DS with dopaminergic agonists promoted a slower rate of heterologous desensitization that diminished responsiveness of the adenylyl cyclase system to dopamine, ACTH, and NaF. Y1DR cells transfected with the dopamine D1 receptor were resistant to the heterologous desensitization of adenylyl cyclase induced by dopaminergic agonists. These latter observations suggest that the pathways of homologous desensitization and heterologous desensitization converge at a common point in the desensitization pathway defined by the DR mutation in Y1 cells.     

Regulation of adenylyl cyclase activity by beta-adrenergic agonists in a desensitization-resistant mutant cell line.

Mutant clones resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were previously isolated from the Y1 mouse adrenocortical tumor cell line. In this study, both parental Y1 cells (Y1DS) and a Y1DR mutant were transfected with a gene encoding the mouse beta 2-adrenergic receptor, and transfectants isolated from both Y1DS and Y1DR cells were shown to express beta 2-adrenergic receptors. These transfectants responded to the beta-adrenergic agonist isoproterenol with increases in adenylyl cyclase activity and steroidogenesis and changes in cell shape. The transfectants were analyzed to determine whether the Y1DR mutation was specific for ACTH-induced desensitization of adenylyl cyclase or also affected desensitization of adenylyl cyclase via the beta 2-adrenergic receptor. Treatment of intact Y1DS transfectants with isoproterenol caused a rapid desensitization of the adenylyl cyclase system to further stimulation by the beta-adrenergic agonist. Treatment of intact cells with isoproterenol did not affect ACTH-stimulated adenylyl cyclase activity, indicating that desensitization was agonist specific or homologous. Y1DR transfectants were resistant to the desensitizing effects of isoproterenol in intact cells as well as in cell homogenates. These results indicate that the mutation in Y1DR transfectants affects a component that is common to the pathways of isoproterenol-induced desensitization and ACTH-induced desensitization of adenylyl cyclase. As determined using the hydrophilic beta-receptor antagonist CGP-12177, isoproterenol caused a rapid sequestration of cell surface receptors in both Y1DS and Y1DR transfectants. From these results we infer that the DR phenotype does not arise from mutations affecting receptor sequestration and that receptor number does not limit the response to isoproterenol in these transfectants.     

Clonal variation in response to adrenocorticotropin in cultured bovine adrenocortical cells: relationship to senescence

During cellular senescence, non-clonal cultures of bovine adrenocortical cells show a continuous decline in the rate of production of cyclic AMP (cAMP) stimulated by adrenocorticotropin (ACTH), without changes in the rate of forskolin- or prostaglandin E1-stimulated cAMP production. We investigated the possible mechanisms for loss of response to ACTH by examining the properties of clones of bovine adrenocortical cells. ACTH-stimulated cAMP production rates were measured in clones immediately after isolation, during long-term growth following isolation, and after subcloning. ACTH-stimulated rates were compared with cAMP production in response to forskolin, which acts directly on the catalytic subunit of adenylate cyclase. The results show that cloning is not necessarily associated with a loss of response to ACTH, but that clones with high ACTH response can give rise to subclones with low response. Clones of adrenocortical cells, at the same approximate population doubling level (PDL), showed ACTH response levels that ranged from 12 to 135 pmol cAMP/10(6) cells/min, whereas mass cultures at this PDL showed approximately 50 pmol/10(6) cells/min. Forskolin-stimulated cAMP production rates in clones varied only over the range of 59-119 pmol/10(6) cells/min and showed no correlation with the ACTH-stimulated rates. All clones were adrenocortical cells, as shown by mitogenic response to angiotensin II and cAMP-inducible 17 alpha-hydroxylase activity. The replicative potential of clones varied widely, and there was no apparent correlation between ACTH response levels and growth potential. The level of ACTH response in each clone was stable during proliferation through at least 25 PD beyond the stage at which the clone was isolated. When clones were subcloned, a clone with a high ACTH response level produced sister subclones that had ACTH response levels ranging from 3% of that of the parent clone to a level slightly greater than that of the parent clone. The growth potential of sister subclones varied widely, as for the parent clones, and there was no obvious correlation between growth potential and ACTH response. Two subclones were cloned; in sub-subclones, levels of ACTH response were again different from each other and also from the parent subclone; in one case, the level of ACTH response was approximately eight-fold higher than that of the parent subclone. These experiments show that clonal variation in the extent of expression of a differentiated property may occur in a normal differentiated cell in culture. The loss of ACTH response and the loss of proliferative potential appear to be independent stochastic events.     

Forskolin-resistant Y1 mutants harbor defects associated with the guanyl nucleotide-binding regulatory protein, Gs

The properties of the adenylate cyclase from forskolin-resistant mutants of Y1 adrenocortical tumor cells was compared with the properties of the enzyme from parental Y1 cells in order to localize the site of mutation. In parental Y1 cells, forskolin stimulated adenylate cyclase activity with kinetics suggestive of an interaction at two sites; in mutant cells, forskolin resistance was characterized by a decrease in enzymatic activity at both sites. Forskolin potentiated the enzyme's responses to NaF and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) in parent and mutant clones, and the mutant enzyme showed the same requirements for Mg2+ and Mn2+ as did the parent enzyme. The adenylate cyclase associated with forskolin-resistant mutants was insensitive to ACTH and was less responsive to Gpp(NH)p than was the parent enzyme. In parental Y1 cells and in the forskolin-resistant mutants, cholera toxin catalyzed the transfer of [32P]ADP-ribose from [32P]NAD+ into three membrane proteins associated with the alpha subunit of Gs; however, the amount of labeled ADP-ribose incorporated into mutant membranes was reduced by as much as 70%. Both parent and mutant membranes were labeled by pertussis toxin to the same extent. The insensitivity of the mutant adenylate cyclase to ACTH and Gpp(NH)p and the selective resistance of the mutant membranes to cholera toxin-catalyzed ADP-ribosylation suggest that a specific defect associated with Gs is involved in the mutation to forskolin resistance in Y1 cells.     

Dynamics of adenylate cyclase desensitization during long-term exposure to the hormone

The dependence of adrenal gland adenylate cyclase desensitization on the dose of in vivo injected ACTH, the time of occurrence and duration of the enzyme refractory period and the dependence of desensitization on the number of ACTH injections were analyzed. The experiments were carried out on guinea pigs injected with prolonged action preparations of ACTH (4 and 6 units) daily for 1-6 days. Intramuscular injections of ACTH caused adenylate cyclase refraction to the repeated action of the hormone. The effect of desensitization was the most conspicuous within the first few hours after hormone injection. The decrease of adenylate cyclase sensitivity and the duration of this effect were found to depend on the ACTH dose as well as on the number of injections. It has been shown for the first time that a single in vivo injection of 0.9% NaCl causes short-term desensitization of adenylate cyclase to the repeated action of much higher doses of ACTH in vitro, presumably due to endogenous ACTH release in response to weak stress exposure. The periodicity of changes in adenylate cyclase sensitivity upon prolonged hormone administration is discussed. Sensitization of the enzyme upon daily short-term exposure to physiological doses of ACTH (administration of 0.9% NaCl for 6 days) was revealed.     

Adrenocorticotropin-induced unresponsiveness in cultured adrenal tumor cells.

Our results demonstrate that adrenocorticotropin (ACTH)-induced refractoriness occurs in cultured adrenal tumor cells. Cells became 85% refractory to ACTH-induced cyclic AMP formation in 20 min and the effect persisted if the hormone remained in the incubation medium. Refractory cells gradually regained hormone-specific responsiveness within 24 h if cultures were incubated in fresh media containing serum. The observed effect is hormone specific since cyclic AMP could not induce unresponsiveness to ACTH. The addition of ACTH plus inhibitors of protein synthesis partially reversed hormone-specific refractoriness. However, preincubation with cycloheximide or diphtheria toxin led to superinduction of ACTH-induced cyclic AMP formation. These experiments suggest that unresponsiveness, following hormonal activation of adrenal cells, may be related to a decrease in hormone-specific binding sites or to synthesis of an adenylate cyclase inhibitor.     

Adrenocorticotropin-induced unresponsiveness in cultured adrenal tumor cells

Our results demonstrate that adrenocorticotropin (ACTH)-induced refractoriness occurs in cultured adrenal tumor cells. Cells became 85% refractory to ACTH-induced cyclic AMP formation in 20 min and the effect persisted if the hormone remained in the incubation medium. Refractory cells gradually regained hormone-specific responsiveness within 24 h if cultures were incubated in fresh media containing serum. The observed effect is hormone specific since cyclic AMP could not induce unresponsiveness to ACTH. The addition of ACTH plus inhibitors of protein synthesis partially reversed hormone-specific refractoriness. However, preincubation with cycloheximide or diphtheria toxin led to superinduction of ACTH-induced cyclic AMP formation. These experimens suggest that unresponsiveness, following hormonal activation of adrenal cells may be related to a decrease in hormone-specific binding sites or to synthesis of an adenylate cyclase inhibitor.     

Desensitization of beta-adrenergic stimulated adenylate cyclase in turkey erythrocytes.

Desensitization of catecholamine stimulated adenylate cyclase (AC) activity is demonstrated in membranes derived from turkey erythrocytes pre-treated with isoproterenol. Membranes from desensitized cells had a loss in maximal catecholamine stimulated adenylate cyclase activity of 104 +/- 13 (pmols/mg protein/10', p less than .001) compared with controls. When adenylate cyclase was maximally stimulated with NaF or Gpp(NH)p, the decrements were 84 +/- 19 (p less than .005) and 92 +/- 32 (p less than .05) pmol/mg protein/10' respectively. There was no change in beta-adrenergic receptor number in membranes derived from treated cells. While the molecular mechanism accounting for the desensitization is uncertain, the data is consistent with the hypothesis that there is a lesion distal to the beta-adrenergic receptor, possibly involving the nucleotide site or the catalytic subunit of adenylate cyclase, causing the desensitization in the isoproterenol treated cells.     

Beta-adrenergic receptor desensitization of wild-type but not cyc lymphoma cells unmasked by submillimolar Mg2+

Treatment with low physiological concentrations of epinephrine (5-50 nM) rapidly desensitizes beta-adrenergic stimulation of cAMP formation in S49 wild-type (WT) lymphoma cells. Previous attempts to detect this early phase of desensitization in cell-free assays of adenylate cyclase (EC 4.6.1.1) after intact cell treatment were unsuccessful. We have now found that reducing the Mg2+ concentrations in the adenylate cyclase assays to less than 1.0 mM unmasked this rapid phase of desensitization of the WT cells, and that high Mg2+ concentrations (5-10 mM) largely obscured the desensitization. Submillimolar Mg2+ conditions also revealed a two- to threefold decrease in the affinity of epinephrine binding to the beta-adrenergic receptor after desensitization with 20 nM epinephrine. Detection of 4 beta-phorbol 12-myristate 13-acetate (PMA) desensitization of the WT beta-adrenergic receptor was also dependent on low Mg2+ as measured either by the decrease in epinephrine stimulation of adenylate cyclase or by the reduction in the affinity of epinephrine binding. Unexpectedly, when cyc- cells were pretreated with 50 nM epinephrine, the beta-adrenergic stimulation of reconstituted adenylate cyclase was not desensitized. The characteristics of the Mg2+ effect on epinephrine- and PMA-induced desensitizations suggest a similar mechanism of action with the most likely events being phosphorylations of the beta-adrenergic receptors. Our data indicate that cAMP-dependent protein kinase (EC 2.7.1.37) may play a role in the desensitization caused by low epinephrine concentrations inasmuch as this phase of desensitization did not occur in the cyc-. For the PMA-induced desensitization, the phosphorylation may be mediated by protein kinase C (EC 2.7.1.37).     

Mechanisms of action of corticotropin-releasing factor and other regulators of corticotropin release in rat pituitary cells

The role of cyclic AMP in the stimulation of corticotropin (ACTH) release by corticotropin-releasing factor (CRF), angiotensin II (AII), vasopressin (VP), and norepinephrine (NE) was examined in cultured rat anterior pituitary cells. Synthetic CRF rapidly stimulated cyclic AMP production, from 4- to 6-fold in 3 min to a maximum of 10- to 15-fold at 30 min. Stimulation of ACTH release by increasing concentrations of CRF was accompanied by a parallel increase in cyclic AMP formation, with ED50 values of 0.5 and 1.3 nM CRF for ACTH and cyclic AMP, respectively. A good correlation between cyclic AMP formation and ACTH release was also found when pituitary cells were incubated with the synthetic CRF(15-41) fragment, which displayed full agonist activity on both cyclic AMP and ACTH release with about 0.1% of the potency of the intact peptide. In contrast, the CRF(21-41) and CRF(36-41) fragments were completely inactive. The other regulators were less effective stimuli of ACTH release and caused either no change in cyclic AMP (AII and VP) or a 50% decrease in cyclic AMP (NE). Addition of the phosphodiesterase inhibitor, methylisobutylxanthine, increased the sensitivity of the ACTH response to CRF but did not change the responses to AII, VP, and NE. In pituitary membranes, adenylate cyclase activity was stimulated by CRF in a dose-dependent manner with ED50 of 0.28 nM, indicating that the CRF-induced elevation of cyclic AMP production in intact pituitary cells is due to increased cyclic AMP biosynthesis. The intermediate role of cyclic AMP in the stimulation of ACTH release by CRF was further indicated by the dose-related increase in cyclic AMP-dependent protein kinase activity in pituitary cells stimulated by CRF with ED50 of 1.1 nM. These data demonstrate that the action of CRF on ACTH release is mediated by the adenylate cyclase-protein kinase pathway and that the sequence requirement for bioactivity includes the COOH-terminal 27 amino acid residues of the molecule. The other recognized regulators of ACTH release are less effective stimuli than CRF and do not exert their actions on the corticotroph through cyclic AMP-dependent mechanisms.     

Desensitization of β-Adrenergic Receptor-Coupled Adenylate Cyclase in Cerebral Cortex After In Vivo Treatment of Rats with Desipramine

Continuous treatment (1-10 days) of rats with desipramine (10 mg/kg, twice per day) caused desensitization of the beta-adrenergic receptor-coupled adenylate cyclase system of cerebral cortical membranes. The decrease in the isoproterenol-stimulated adenylate cyclase activity was more rapid and greater than the decrease in the number of beta-adrenergic receptors in membranes during treatment of the membrane donor rats with desipramine, indicating that the desensitization occurring at an early stage of the treatment was not accounted for solely by the decrease in the receptor number. Neither the guanine nucleotide regulatory protein (N) nor the adenylate cyclase catalyst was impaired by the drug treatment, since there was no decrease in the cyclase activity measured in the presence or absence of GTP, guanyl-5'-yl-beta-gamma-imidodiphosphate [Gpp(NH)p], NaF, or forskolin. Gpp(NH)p-induced activation of membrane adenylate cyclase developed with a lag time of a few minutes in membranes from control or drug-treated rats. The lag was shortened by the addition of isoproterenol, indicating that beta-receptors were coupled to N in such a manner as to facilitate the exchange of added Gpp(NH)p with endogenous GDP on N. This effect of isoproterenol rapidly decreased during the drug treatment of rats. Thus, functional uncoupling of the N protein from receptors was responsible for early development of desensitization of beta-adrenergic receptor-mediated adenylate cyclase in the cerebral cortex during desipramine therapy.     

ACTH-induced refractoriness in cultured adrenal cell line (Y1).

Treatment of cultured mouse adrenal cells Y₁ with ACTH induced cell refractoriness to further hormonal stimulation. When ACTH was added to the cells every 2 hours the first addition increased the levels of 20αOH-progesterone and cAMP secreted into the medium. Upon the second and third additions of ACTH the levels of 20αOH-progesterone and cAMP secreted were greatly diminished and upon the fourth addition of ACTH were absent. Prolonged incubation (14 hours) with different concentrations of ACTH (5 × 10^?11 M to 10^?6 M) induced a dose-related steroidogenic refractoriness to further ACTH stimulation, 10^?8 M ACTH inducing complete refractoriness. The number of ACTH binding sites of cell particles prepared from desensitized cells was similar to that of the control but ACTH failed to stimulate the adenylate cyclase of desensitized cells, whereas the enzyme responded fully to NaF and Gpp(NH)p. The cAMP phosphodiesterase activity was similar in both desensitized and control cells. In addition the steroidogenic response to dibutyryl cAMP of desensitized cells was abolished. Thus, ACTH-induced adrenal cell desensitization seems to be related to at least two phenomena : a defect in the “coupling” between the hormone-receptor sites and the adenylate cyclase and an alteration of certain steps beyond cAMP formation.     

Hormonal activation of the cAMP-dependent protein kinases in AtT20 cells. Preferential activation of protein kinase I by corticotropin releasing factor, isoproterenol, and forskolin

The hormonal regulation of adenylate cyclase, cAMP-dependent protein kinase activation, and adrenocorticotropic hormone (ACTH) secretion was studied in AtT20 mouse pituitary tumor cells. Corticotropin releasing factor (CRF) stimulated cAMP accumulation and ACTH release in these cells. Maximal ACTH release was seen with 30 nM CRF and was accompanied by a 2-fold rise in intracellular cAMP. When cells were incubated with both 30 nM CRF and 0.5 mM 3-methylisobutylxanthine (MIX) cAMP levels were increased 20-fold, however, ACTH release was not substantially increased beyond release seen with CRF alone. The activation profiles of cAMP-dependent protein kinases I and II were studied by measuring residual cAMP-dependent phosphotransferase activity associated with immunoprecipitated regulatory subunits of the kinases. Cells incubated with CRF in the absence of MIX showed concentration-dependent activation of protein kinase I which paralleled stimulation of ACTH release. Protein kinase II was minimally activated. When cells were exposed to CRF in the presence of 0.5 mM MIX there was still a preferential activation of protein kinase I, although 50% of the cytosolic protein kinase II was activated. Complete activation of both protein kinases I and II was seen when cells were incubated with 0.5 mM MIX and 10 microM forskolin. Under these conditions cAMP levels were elevated 80-fold. CRF, isoproterenol, and forskolin stimulated adenylate cyclase activity in isolated membranes prepared from AtT20 cells. CRF and isoproterenol stimulated cyclase activity up to 5-fold while forskolin stimulated cyclase activity up to 15-fold. Our data demonstrate that ACTH secretion from AtT20 cells is mediated by small changes in intracellular levels of cAMP and activation of only a small fraction of the total cytosolic cAMP-dependent protein kinase in these cells is required for maximal ACTH secretion.     

On the mechanism of isoproterenol-induced desensitization of adenylate cyclase in cultured differentiated hepatocytes

The adenylate cyclase of cultured differentiated RL-PR-C hepatocytes is desensitized to 1-isoproterenol by exposure to this beta-agonist. Virtually complete desensitization occurred by 60 min (intact cells) or 30 min (isolated plasma membranes). Isoproterenol was maximally effective at 10 micrometers, although substantial desensitization occurred at isoproterenol concentrations as low as 10 nM. Protein synthesis was not required for desensitization. Recovery from desensitization under tissue culture conditions was only 25% complete by 24 h. Maximum desensitization was accompanied by only a modest 35% decrease in binding sites (as determined by binding assays with [3H]dihydroalprenolol), with no change in binding affinity. Adenylate cyclase desensitized to 1-isoproterenol responded normally to guanine nucleotides and to fluoride, suggesting that the regulatory and catalytic proteins were not the sites of the desensitization "defect'. Using N-ethylmaleiimide to inactive the regulatory and catalytic proteins, and dicyclohexylcarbodiimide to inactivate the beta-adrenergic receptor, of intact hepatocytes, various heterologous cell fusion hybrids were produced, and their adenylate cyclases tested for responsiveness to 1-isoproterenol; only hybrids containing "desensitized' receptor failed to respond to isoproterenol. These results suggest that the mechanism of desensitization to isoproterenol involves only the receptor component of the receptor-regulatory protein(s)-adenylate cyclase complex, and that the receptors are reduced in number and/or ability to interact with the regulatory protein as a result of the desensitization process.     

Challenge of hepatocytes by glucagon triggers a rapid modulation of adenylate cyclase activity in isolated membranes.

Membrane fractions obtained from hepatocytes treated with glucagon exhibited a decreased glucagon (with or without GTP)-stimulated adenylate cyclase activity. A maximum effect was seen in around 5 min. No change in the rate of cyclic AMP production was observed for the basal, NaF-, p[NH]ppG (guanosine 5'-[beta, gamma-imido]-triphosphate)- and GTP-stimulated states of the enzyme. The lag observed in the p[NH]ppG-stimulated adenylate cyclase activity of native membranes was abolished when membranes from glucagon-pretreated cells were used. When Mn2+ replaced Mg2+ in the assays, the magnitude of the apparent desensitization was decreased. Mn2+ abolished the lag of onset of p[NH]ppG-stimulated activity in native membranes. The desensitization process was dose-dependent on glucagon, which exhibited a Ka of 4 X 10(-10) M. Depletion of intracellular ATP did not affect this process. It is suggested that this desensitization occurs at the level of the guanine nucleotide-regulatory protein.     

Evaluation of receptor function in ACTH-responsive and ACTH-insensitive adrenal tumor cells.

Two variant cell lines (Y6 and OS3), derived from the ACTH-sensitive mouse adrenocortical tumor clone Y1, are defective in the ACTH-sensitive adenylate cyclase system. This study further characterizes the nature of the defects in Y6 and OS3 cells using ACTH1-10, ACTH4-10, and cholera toxin. In Y1 cells, ACTH1-39, ACTH1-10, and ACTH4-10 stimulated steroidogenesis to the same maximum level with Kd' values of 5 x 10(-11) M, 5 x 10(-7) M and 10(-4) m respectively. ACTH1-10 (0.4 mM) and ACTH4-10 (3.2 mM) increased the accumulation of adenosine 3',5'-monophosphate (cAMP) in Y1 cells two- to three-fold. Cholera toxin increased steroidogenesis and cAMP accumulation in Y1 cells with Kd' values of 0.4 ng/mL and 9 ng/mL respectively. Y6 and OS3 cells responded to added cholera toxin with increased cAMP accumulation and increased steroidogenesis but did not respond to ACTH1-39, ACTH1-10, or ACTH4-10 at concentrations effective in Y1 cells. These data are interpreted to suggest that Y6 and OS3 cells are defective in a process or component that links the principal binding regions of the ACTH receptor to the catalytic subunit of the adenylate cyclase system. Attempts to were made to assess the interactions of ACTH with the principal binding regions of the ACTH receptor by analysis of binding of radioactive, iodinated ACTH1-24. ACTH binding, however, showed low affinity, high capacity, and no target-tissue specificity, and was considered not to be useful in evaluating the integrity of the ACTH receptor.     

Transmembrane controls in cultured human thyroid carcinoma

It is well known that many of thyroid carcinoma are capable of responding to TSH, but our studies shown that there are some alteration in this responsiveness. The adenylate cyclase responsiveness to TSH was usually greater in thyroid carcinoma than in adjacent histologically normal thyroid tissue. The level of increased response of adenylate cyclase were correlated with the level of enhanced expression of ras oncogene product p21 assessed by Western blotting analysis. The TSH induced desensitization of adenylate cyclase was not observed in some differentiated carcinoma. This loss of desensitization may be reflect the change in ADP-ribosylable Gi protein. In the differentiated carcinoma, the capacity of EGF receptor was higher than that in normal thyroid. The EGF binding to cultured carcinoma cells did not increase in response to TSH. These altered properties of transmembrane control in human thyroid carcinoma may be related to the neoplastic growth.     

GRF is a highly potent activator of adenylate cyclase in the normal human, bovine and rat pituitary: interaction with somatostatin

The effect of GRF adenylate cyclase activation was studied in normal human, bovine and rat pituitary tissues. Human GRF (hGRF) activates adenylate cyclase in normal human pituitary membrane preparations in a concentration dependent manner (ED5 0 = 10(-11) M). In bovine pituitary cells hGRF stimulates GH secretion into the medium (ED5 0 = 7 X 10(-12) M) and activates adenylate cyclase (ED5 0 = 10(-11) M). In normal rat pituitary cells in monolayer culture, rat GRF (rGRF) stimulates adenylate cyclase (ED5 0 = 3 X 10(-11) M). In normal human pituitary membrane preparations and in normal rat pituitary cells in culture, somatostatin inhibits GRF-stimulated adenylate cyclase in a non-competitive manner, while it does not affect basal (i.e. non-stimulated) adenylate cyclase levels. VIP, a peptide which is structurally homologous to hGRF and rGRF is a weak GRF-agonist and activates adenylate cyclase in human and rat pituitary preparations at concentrations greater than 10 nM.