AMP Phosphodiesterase Research Articles

Abstract 5174: Phosphodiesterase 10A inhibition as a novel approach to suppress β-catenin signaling in ovarian cancer cells

Abstract Canonical Wnt/β-catenin signaling is known to be associated with platinum resistance in ovarian cancer in which inhibitors hold promise for the treatment of refractory disease. Phosphodiesterase 10A (PDE10A) is a dual cyclic AMP and cyclic GMP phosphodiesterase isozyme recently implicated in colon cancer. PDE10A inhibition in colon cancer cells by siRNA or small molecule inhibitors increased cGMP levels and activated PKG to inhibit β-catenin signaling. A novel PDE10 inhibitor, ADT-061, was identified by screening a library of indene derivatives, and showed strong antineoplastic activity in the Apc+/min-FCCC mouse (Lee K et al., unpublished data). Cyclic GMP and phosphodiesterases participate in the ovarian follicular development, although little is known about PDE10A expression in ovaries, especially with regard to a potential role in ovarian tumorigenesis. PDE10A protein was found to be expressed in various established ovarian cancer cell lines at higher levels than immortalized or primary ovarian surface epithelial cell lines. Pf-2545920, a known PDE10A inhibitor, and ADT-061 inhibited the growth of multiple ovarian tumor cell lines with IC50s around 20µM and 0.5µM, respectively. Both compounds induced apoptosis after 24h treatment, as measured by PI/Annexin-V staining and PARP cleavage. Pf-2545920 and ADT-061 induced phosphorylation of VASP at Ser157 and Ser239 in various ovarian cancer cell lines, indicating activation of cyclic AMP and cyclic GMP signaling, respectively. Treatment also decreased levels of β-catenin and downstream targets of TCF-dependent transcription, including c-MYC, survivin and cyclin-D1. Homozygous knockout PDE10A clones of OV-90 ovarian cancer cells obtained using CRISPR/Cas9 showed decreased clonogenic potential, decreased Pf-2545920-mediated VASP phosphorylation and β-catenin, c-MYC and survivin expression. Ongoing efforts are focused on the development of more potent ADT-061 analogs. These observations support further study of a role of PDE10 in ovarian tumorigenesis and the development of ADT-061 or analogs for the treatment of refractory ovarian cancer as well as the prevention of malignant recurrence. Citation Format: Luciana Madeira Da Silva, Elaine Gavin, Kevin J. Lee, Veronica Ramírez-Alcántara, Kristy L. Berry, Holly T. Taylor, Alla Musiyenko, Ileana V. Aragon, Adam B. Keeton, Jennifer Scalici, Rodney P. Rocconi, Gary A. Piazza. Phosphodiesterase 10A inhibition as a novel approach to suppress β-catenin signaling in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5174. doi:10.1158/1538-7445.AM2017-5174

Abstract A76: Targeting phosphodiesterase 10A for chemoprevention and treatment of ovarian cancer.

Abstract Phosphodiesterase 10A (PDE10A) is a dual cyclic AMP and cyclic GMP phosphodiesterase that is highly expressed in the brain striatum, testis and thyroid and this restricted expression in peripheral tissue has been clinically exploited for the treatment of schizophrenia and Huntington's disease. Interestingly, our group has recently found that PDE10A is overexpressed in colon, lung and breast tumors when compared to their healthy tissue correlates. Moreover, our studies indicate that PDE10A inhibition in cancer cells by RNA interference or by small molecule inhibitors leads to activation of cGMP/PKG and inhibition of β-catenin signaling pathway that culminates with apoptotic cell death. Here we are investigating the role of PDE10A in ovarian cancer by using genetic approaches (RNA interference and CRISPR/Cas9 genome editing) and novel PDE10A small molecule inhibitors that we generated. We found that PDE10A protein is expressed in various established ovarian cancer cell lines at higher levels than in immortalized ovarian surface epithelial cell lines. Using a known PDE10A inhibitor, Pf-2545920, and novel sulindac derivatives that we generated with high specificity towards the PDE10A isozyme, we observed potent growth inhibition properties (IC50s at low micromolar or sub-micromolar concentrations) for all ovarian cancer cell lines tested. One of our novel PDE10A inhibitors, called MCI-030, which showed IC50~0.5μM for ovarian cancer cells, was orally administered to C57BL/6 mice at a safe dose of 100mg/Kg and revealed a stable concentration of approximately 2μM in the plasma and ovarian tissue for about 8h. Mechanistically, our results showed that PDE10A inhibitors induced apoptosis as measured by PI/Annexin V staining. Pf-2545920 induced phosphorylation of VASP at Ser157 and Ser239 in OV-90, OVCAR3 and SKOV3 cells, indicating activation of cyclic nucleotide signaling pathway. In addition, a decrease in the levels of β-catenin, survivin, c-Myc and cyclin D1 was observed in OV-90 cells treated with Pf-2545920 for 24h. We used CRISPR-Cas9 to delete a 97bp segment in exon 7 of PDE10A in the diploid ovarian cancer cell line OV-90. Homozygous knockout PDE10A clones were obtained and showed decreased clonogenic potential and decreased Pf-2545920-mediated pVASP phosphorylation in vitro. We are currently investigating the expression of PDE10A in clinical specimens of ovarian tumors by qPCR, western-blotting and immunohistochemistry. Our future work includes testing MCI-030 in vivo using xenograft intraperitoneal models of ovarian cancer, as well as in vivo experiments with OV-90 PDE10 knockout cells. Our ultimate goal for the proposed studies of PDE10 as a novel chemoprevention target for ovarian cancer is to develop a potent PDE10 inhibitor with negligible toxicity that can be used as a chemoprevention agent for women at high risk for this malignancy. Citation Format: Luciana Madeira da Silva, Elaine Gavin, Kevin Lee, Ileana Aragon, Veronica Ramírez-Alcántara, Jennifer Scalici, Rodney P. Rocconi, Gary A. Piazza. Targeting phosphodiesterase 10A for chemoprevention and treatment of ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A76.

Correlation between hydrophobicity of N-alkylxanthine derivatives and their biological activities on guinea-pig isolated tracheal smooth muscle.

Cyclic (c) AMP phosphodiesterase (PDE) inhibitory activities of N-alkylxanthine derivatives (3-methyl-,3-ethyl-,3-propyl-,3-butyl-,1,3-dimethyl-,1-methyl-3-ethyl-,1-methyl - 3-propyl- and 1-methyl-3-butyl xanthines) and their relaxant effects on carbachol-induced contraction and on resting tone guinea-pig isolated tracheal smooth muscle have been investigated. The PDE inhibition constant (Ki) and the concentration producing 50% tracheal smooth muscle relaxation in-vitro (EC50) were determined. Significant correlations between the -log Ki values and the -log EC50 values on the carbachol-induced contraction or on the resting tone were found (r = 0.902 and 0.892). The apparent partition coefficient (P) between n-octanol and pH 7.4 phosphate-buffered saline (PBS) was measured as an index of hydrophobicity of the xanthine derivatives. There were significant correlations between log P and both -log EC50 values and between the log P and -log Ki values. These findings suggest that the cAMP PDE inhibitory activity of N-alkylxanthine derivatives contributes to the mechanism of bronchodilatory action, and that an increase in hydrophobicity of the xanthine molecule enhances the biological activity.

Active and energy-dependent rapid formation of cell aggregates in the thermophilic photosynthetic bacterium Chloroflexus aggregans

The thermophilic filamentous phototroph Chloroflexus aggregans was able to form a bacterial mat-like dense cell aggregate rapidly. The aggregate formation, which was observed in growing cells in a liquid medium in a bottle, occurred every time within 20–30 min after the cells were dispersed by shaking. The aggregation depended on the energy supplied by photosynthesis or respiration. Cells aggregated most rapidly under temperature and pH conditions that support maximum growth. The aggregation was also accelerated by the addition of 3-isobutyl-1-methylxanthine that inhibits cyclic 3′,5′-AMP phosphodiesterase. Microscopic observation revealed that the bacterium has a fast gliding mobility (1–3 μm s −1). The distinctive cell aggregation of C. aggregans was due to this rapid gliding movement.

Active and energy-dependent rapid formation of cell aggregates in the thermophilic photosynthetic bacterium Chloroflexus aggregans.

The thermophilic filamentous phototroph Chloroflexus aggregans was able to form a bacterial mat-like dense cell aggregate rapidly. The aggregate formation, which was observed in growing cells in a liquid medium in a bottle, occurred every time within 20-30 min after the cells were dispersed by shaking. The aggregation depended on the energy supplied by photosynthesis or respiration. Cells aggregated most rapidly under temperature and pH conditions that support maximum growth. The aggregation was also accelerated by the addition of 3-isobutyl-1-methylxanthine that inhibits cyclic 3',5'-AMP phosphodiesterase. Microscopic observation revealed that the bacterium has a fast gliding mobility (1-3 microm s(-1)). The distinctive cell aggregation of C. aggregans was due to this rapid gliding movement.

Species differences in localization of cardiac cAMP-phosphodiesterase activity: a cytochemical study.

The localization of the membrane-bound cyclic 3',5'-AMP phosphodiesterase in cardiac tissues of both, rat and dog was studied by cytochemical method. 40 microm thick slices from glutaraldehyde fixed heart tissue were incubated in the medium with cAMP as a substrate and Pb ions as a capture metal of the reaction product. The cAMP-PDE activity in the rat ventricle was only shown positive on the sarcolemma. Whereas, in canine ventricular tissue the cAMP-PDE activity in cardiomyocytes was shown on the sarcolemma, on the junctional sarcoplasmic reticulum and on subsarcolemmal cisternae. The results confirm differences in the localization of cAMP-PDE in dog and rat heart.

Cross-talk between glucagon- and adenosine-mediated signalling systems in rat hepatocytes: effects on cyclic AMP-phosphodiesterase activity.

The effect of adenosine analogues on glucagon-stimulated cyclic AMP accumulation in rat hepatocytes was explored. N6-Cyclopentyladenosine (CPA), 5'-N-ethylcarboxamidoadenosine and N6-(R-phenylisopropyl)adenosine inhibited in a dose-dependent manner the cyclic AMP accumulation induced by glucagon. This effect seems to be mediated through A1 adenosine receptors. Pertussis toxin completely abolished the effect of CPA on glucagon-stimulated cyclic AMP accumulation in whole cells which suggested that a pertussis-toxin-sensitive G-protein was involved. On the other hand, this action of adenosine analogues on glucagon-induced cyclic AMP accumulation was reverted by the selective low-Km cyclic AMP-phosphodiesterase inhibitor Ro 20-1724. Analysis of cyclic AMP-phosphodiesterase activity in purified hepatocyte plasma membranes showed that glucagon in the presence of GTP inhibited basal PDE activity by 45% and that CPA reverted this inhibition in dose-dependent manner. In membranes derived from pertussis-toxin-treated rats, we observed no inhibition of cyclic AMP-phosphodiesterase activity by glucagon in the absence or presence of CPA. Our results indicate that in hepatocyte plasma membranes, stimulation of adenylate cyclase activity and inhibition of a low-Km cyclic AMP phosphodiesterase activity are co-ordinately regulated by glucagon, and that A1 adenosine receptors can inhibit glucagon-stimulated cyclic AMP accumulation by blocking glucagon's effect on phosphodiesterase activity.

A reverse-phase HPLC method for cyclic nucleotide phosphodiesterases activity and classification.

Cyclic AMP and cyclic GMP phosphodiesterases (PDEs) exist as a family of isoenzymes which possess distinct primary amino acid sequences and are encoded by distinct genes (Beavo & Refsnyder, 1990). These multiple enzymes have been classified into five major groups according to kinetic, physical, immunological and regulatory properties (Beavo and Refsnyder, 1990). The Phosphodiesterase plays a pivotal role in the cellular signalling process, limiting the cellular response to agents which utilize cyclic nucleotides as second messengers. However, these isoenzymes do not solely act as a brake on cyclic nucleotide synthesis, since their activity states are also regulated by a variety of hormones such as insulin, adrenaline, glucagon, Prostaglandin I2 and Prostaglandin E1 (Zinman & Hollenberg, 1974; Loten et al., 1978; Makino& Kono, 1980; Heyworth et al., 1983; Macphee et al., 1988). Cyclic AMP plays a dominant role in the neuro-hormonal regulation of cardiac contractility (Hartzell,1988; Lindemann and Watanabe,1989).

Changes of calmodulin concentration and cyclic 3′, 5′-nucleotide phosphodiesterase activities in cardiac muscle of hyper- and hypothyroid rats

To investigate the effect of thyroid hormone on cardiac muscle dysfunction in hyper- and hypothyroid states, we evaluated cyclic 3',5'-nucleotide metabolism by measuring cyclic 3',5'-nucleotide phosphodiesterase activity and calmodulin concentrations in the cardiac muscles of hyper- and hypothyroid rats. Cyclic AMP (cAMP) concentration was significantly high in the cardiac muscle of hyperthyroid rats and low in that from hypothyroid rats compared with control rats. Cyclic AMP and cyclic GMP phosphodiesterase activities were significantly decreased in the soluble fraction of cardiac muscle from hyperthyroid rats and markedly increased in this fraction in hypothyroid rats compared with normal animals. Calmodulin concentration was high in hyperthyroid and low in hypothyroid rats. It was concluded from these findings that low cAMP-phosphodiesterase activity might, in part, bring about the high concentration of cAMP. Calmodulin was significantly high in the cardiac muscle of hyperthyroid rats and the reverse was the case in hypothyroid rats compared with normal rats. The implication is that, in hyper- and hypothyroid states, these changes may play an important role in cardiac function via their effect on cyclic nucleotide and Ca2+ metabolism.

Differential hemodynamic responses to selective inhibitors of cyclic nucleotide phosphodiesterases in conscious rats.

Selective inhibition of either the low Km cyclic AMP (cAMP) or low Km cyclic GMP (cGMP) phosphodiesterase (PDE) promotes vasorelaxation and, consequently, produces depressor effects. To evaluate the systemic and regional hemodynamic effects of selective inhibitors of these PDE isozymes, CI-930 (0.1-10 mg/kg), an inhibitor of low Km cAMP PDE, or zaprinast (3-30 mg/kg), an inhibitor of low Km cGMP PDE, was given i.v. to conscious, normotensive rats. The rats were chronically instrumented with vascular catheters and either an ultrasonic transit-time flow probe around the ascending aorta or miniaturized pulsed Doppler flow probes around the superior mesenteric and left renal arteries and the abdominal aorta. CI-930 and zaprinast, at cumulative doses of 3 and 30 mg/kg, respectively, produced comparable reductions in mean arterial pressure (-22 +/- 3 and -19 +/- 4 mm Hg, respectively) and total peripheral resistance (-0.41 +/- 0.07 and -0.42 +/- 0.06 mm Hg/ml/min, respectively) but affected other hemodynamic variables differently. CI-930 at 3 mg/kg increased the heart rate (HR), maximal aortic flow acceleration (dF/dt), and peak aortic flow and decreased the stroke volume (SV). Cardiac output (CO) was not affected by CI-930. Zaprinast at 30 mg/kg increased the CO, dF/dt, and peak aortic blood flow. The HR and SV were unaffected by zaprinast. Although both CI-930 and zaprinast increased the dF/dt and peak aortic flow, these parameters were affected more by CI-930 than by zaprinast. CI-930 decreased hindquarter, mesenteric, and renal vascular resistances in a dose-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylate cyclase system of human trichilemmoma cell line

The adenylate cyclase system of an established human trichilemmoma cell line was investigated. Stimulators of human epidermal adenylate cyclase system, epinephrine, histamine, adenosine, and prostaglandin E increased cyclic AMP levels of the trichilemmoma cells. The effects of epinephrine, histamine, and adenosine were inhibited by the addition of propranolol (a beta-adrenergic antagonist), cimetidine (histamine H2-antagonist), and theophylline (adenosine-receptor antagonist), respectively. The epinephrine, histamine, and prostaglandin E effects were augmented by the addition of cyclic AMP (cAMP) phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX); the adenosine effect was augmented by another phosphodiesterase inhibitor, papaverine. Without the addition of these phosphodiesterase inhibitors, the maximal accumulations were observed at 3 min incubation. Following this, the cAMP content returned to the basal level, and the cells did not respond to repeated stimulations with the same initial stimulator. This fact indicates receptor-specific refractoriness. For example, epinephrine-pretreated cells did not respond to epinephrine, but retained their sensitivity to histamine. It has been known that normal epidermal keratinocytes are regulated in vitro by glucocorticoids, colchicine, and retinoids, resulting in the augmentation of their beta-adrenergic response. Only hydrocortisone treatment on the trichilemmoma cells resulted in the augmentation of the beta-adrenergic response. Although the established human trichilemmoma cell line has similar adenylate cyclase systems as normal epidermis, it apparently has lost some of the regulatory mechanism of the beta-adrenergic response.

Dimethyl sulfoxide-induced augmentation of adenosine-adenylate cyclase response of pig skin epidermis

Adenosine-adenylate cyclase response in pig skin epidermis showed a specific increase after long-term (24 h) incubation in the presence of 0.5%-1% dimethyl sulfoxide (DMSO). There was no significant difference between control and DMSO-treated epidermis with regard to cyclic AMP (cAMP) phosphodiesterase activity. DMSO had no effect on the basal cAMP levels of epidermis; beta-adrenergic and histamine-adenylate cyclase responses were not affected. The direct addition of DMSO at the time of incubation with various adenylate cyclase stimulators (adenosine, epinephrine, and histamine) had no effect on agonist-induced cAMP accumulation effects. It was concluded that DMSO affected epidermal keratinocytes during long-term incubation, resulting in a specific increase in the adenosine-adenylate cyclase response. Although the biological significance of this DMSO effect remains to be determined, it should be kept in mind when using DMSO as a solvent for various chemicals in the experiments dealing with epidermal keratinocytes in vitro.

Characterization of cyclic AMP and cyclic GMP phosphodiesterases in Tetrahymena cilia

Abstract 1. 1. The activities of cyclic AMP and cyclic GMP phosphodiesterases were found to be associated with both soluble and particulate fractions in Tetrahymena cilia. 2. 2. The properties of these enzymes in ciliary fractions were similar to those of cell bodies in K m , pH dependency, inhibition by methylxanthine and lack of a requirement for Mg2+. 3. 3. These enzyme activities of cell bodies of behavioral mutants were higher than those of wild type, while the enzyme activities of cilia did not show differences in both wild and mutant cells.

125 Cyclic-3′-5′-AMP phosphodiesterases in a spontaneous mammary tumour in the mouse

125 Cyclic-3′-5′-AMP phosphodiesterases in a spontaneous mammary tumour in the mouse

Cytochemical demonstration of cyclic 3', 5' AMP phosphodiesterase in different tissue of migratory locust (Locusta migratoria migratorioides R.F.).

The localization of cyclic 3', 5'-AMP phosphodiesterase was studied by ultrastructural cytochemical methods in the various tissues of Locusta migratoria. Large amounts of electron dense, granular reaction products were detectable on the surface of the corpora allata and the corpus cardiacum, bound to the basal lamina. In the protocerebral neuropile rather large amounts of reaction products were observed in the processes of the glial cells. A significant number of lead phosphate deposit was found to occur on the membrane of certain large axons, the microtubules of the axons, furthermore on the membrane of several terminals. Reaction product was also observable in certain terminals, bound to the synaptic vesicles and the mitochondria. At the same time, electron dense deposits were not detectable at all on the surface of cerebral neurons. In the case of myocardium, reaction product was only found on the basal lamina and the extracellular surface of the sarcolemma. On the basis of our results it can be stated that the cyclic 3', 5'-AMP phosphodiesterase is detectable by cytochemical methods in different tissues of Locusta migratoria and presumably it fulfils the task of the extracellular cAMP level regulation.

Measurements of cyclic AMP and cyclic GMP phosphodiesterase activity in isolated tubular segments

The structural and functional heterogeneity of the renal tubular system is well recognized. To date however, the biochemical steps in the mechanism of action of hormones whose effects are mediated by cyclic 3',5'-AMP (cAMP) have been studied primarily in the whole kidney or in major anatomic subdivisions (cortex, medulla, and papilla) of mammalian renal tissue [1, 2]. Recently, however, Imbert, Chabardes, and Morel introduced a micromethod for the assay of adenylate cyclase activity in specific nephron segments [3, 4] and demonstrated that adenylate cyclase stimulated by vasopressin (VP), parathyroid hormone, calcitonin, or isoproterenol is limited to well-defined segments of the renal tubular system [5]. Stimulation of adenylate cyclase, although important, is only the initial step in the sequence of intracellular biochemical events leading to the ultimate functional response of the tubule to a hormone. The rate of cAMP accumulation and the intracellular cAMP concentration are dependent not only on the rate of biosynthesis but also on the rate of hydrolysis by a specific enzyme, cyclic 3',5'-AMP phosphodiesterase (cAMP-PDIE), and on the rate of efflux of cAMP from the cell [6]. Our previous studies [7] indicated that the distribution of enzymes of cAMP and cyclic 3',5'-GMP (cGMP) metabolism were different in glomeruli compared to mixed cortical tubular fractions or to cortical slices, suggesting that the catabolism of cyclic nucleotides differs in specific substructures of the nephron.

Cyclic 3′,5′-AMP phosphodiesterase in Physarum polycephalum: II. Kinetic properties

Some kinetic characteristics of soluble and particulate cyclic 3′,5′-AMP phosphodiesterases from Physarum polycephalum are presented. The nature of enzyme inhibition by various agents is reported. The relationship between various enzyme inhibitors and their reported chemotactic properties in Physarum is examined. The results suggest that the chemoattractant effect of several inhibitors may be related to their ability to inhibit the particulate and extracellular phosphodiesterases, but is unrelated to inhibition of the intracellular enzyme.

Cyclic 3′,5′-AMP phosphodiesterase in Physarum polycephalum I. Chemotaxis toward inhibitors and cyclic nucleotides

The effect of several inhibitors of the enzyme cyclic 3′,5′-AMP phosphodiesterase as chemoattractants in Physarum polycephalum was examined. Of the compounds tested, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Roche 20-1724/001) and 1-ethyl-4-(isopropylidinehydrazino)-1H-pyrazolo-(3,4- b)-pyridine-5-carboxylic acid ethyl ester, hydrochloride (Squibb 20009) were the most potent attractants. 3-Isobutyl-1-methyl xanthine, theophylline, and morin (a flavanoid) were moderate attractants and sometimes gave negative chemotaxis at high concentrations. Cyclic 3′,5′-AMP was an effective, but not potent attractant. A repellent effect following the positive chemotactic action was sometimes observed with cyclic 3′,5′-AMP at concentrations as high as 1 · 10 −2 M. Dibutyryl cyclic AMP appeared to be a somewhat more potent attractant than cyclic 3′,5′-AMP. The 8-thiomethyl and 8-bromoderivatives of cyclic AMP, which are poorly hydrolyzed by the phosphodiesterase, were not attractants in Physarum. Possible participation of cyclic 3′,5′-AMP in the directional movement in P. polycephalum is discussed.

Inhibition of 3',5'–AMP Phosphodiesterase by Biflavonoids and Xanthones

Inhibition of 3',5'–AMP Phosphodiesterase by Biflavonoids and Xanthones

The adenylate cyclase-cyclic AMP-phosphodiesterase system in pathological human thyroid

The adenylate cyclase-cyclic AMP-phosphodiesterase system of human thyroid tissues adjacent to cold nodules (control), two follicular adenomas, one hyperplastic thyroid and one hyperfunctioning follicular carcinoma have been compared. In the hyperfunctional follicular carcinoma the basal adenylate cyclase is much higher than in control tissue, carcinoma adenylate cyclase does not respond to TSH and prostaglandin E1, whereas it responds normally to fluoride. In the hyperplastic, but hypofunctional thyroid the basal adenylate cyclase is higher than in normal tissue whereas the response to TSH, PGE1, and fluoride is normal. No difference between the follicular adenomas and normal thyroid stimulated and unstimulated adenylate cyclase was observed. Furthermore in various thyroid tissues no changes in the level of cyclic AMP phosphodiesterase was found. Our data indicate a greater change in the synthesis rather than in degradation of cyclic AMP in the human pathological thyroids studied.