Activation of cyclic AMP-dependent protein kinase(s) by growth hormone in the liver and adrenal gland of the rat.

A single dose of growth hormone (10 mg/kg, i.p.) was injected into male weanling rats (50--60 g), and the temporal changes in cyclic AMP concentration, protein kinase activation, and ornithine decarboxylase activation were measured in the liver and adrenal gland. The level of cyclic AMP did not change significantly from control values in either liver or adrenal following growth hormone administration. Cyclic AMP-dependent protein kinase(s); however, was markedly activated in liver and adrenal within 30 min. Protein kinase remained activated for more than 4 hr in the liver, while activation of protein kinase in the adrenal returned to control value within 2 hr. Ornithine decarboxylase activity was elevated 20-fold in liver within 4 hr of injection and was increased 7- to 8-fold in be adrenal within l hr. These observations are discussed with regard to the generality of the role of cyclic AMP as the second messenger for target-specifici trophic hormone action and the significance of protein kinase activiation as an index of the cyclic nucleotide involvement in the growth response.     

Induction of ornithine decarboxylase by biliverdin in rat liver.

The administration of biliverdin (0.1mg/g of body weight) into the peritoneal cavity of rats resulted in the induction of ornithine decarboxylase in the liver. When the temporal relationships between the changes in intracellular adenosine 3', 5'-cyclic monophosphate (cyclic AMP) level, cyclic AMP-dependent protein kinase activity and the induction of ornithine decarboxylase were investigated, the concentration of cyclic AMP increased significantly 2 h after the administration of biliverdin, while cyclic AMP-dependent protein kinase was activated after 2–4 h. The hepatic ornithine decarboxylase activity began to increase 4 h after biliverdin injection. These results suggest that there is some sequential relationship between the increase of cyclic AMP, the activation of cyclic AMP-dependent protein kinase and the induction of ornithine decarboxylase although the direct correlation of these three events remains to be elucidated.     

Opiates and cultured neuroblastoma x glioma cells. Effect on cyclic AMP and polyamine levels and on ornithine decarboxylase and protein kinase activities.

In cultured NG 108-15 neuroblastoma x glioma cells, opiates decreased cellular cyclic AMP and polyamine levels. This decrease was related to the inhibition of ornithine decarboxylase and cyclic AMP-dependent protein kinase activities during the acute exposure of the cells to the drugs. Growing the cells in the presence of opiates for several days led to drug addiction. In the tolerant-addicted cells, polyamine and cyclic AMP levels were close to normal values as were the activities of ornithine decarboxylase and cyclic AMP-dependent protein kinase. Removal of the opiate from 'addicted' cells, by either washing or by adding the antagonist naloxone, resulted in an increase in cyclic AMP and polyamine levels and the activities of ornithine decarboxylase and cyclic AMP-dependent protein kinase. The effect of opiates was closely related to their biological activities. Inactive enantiomorphs did not affect cyclic AMP or polyamine levels; neither did they decrease ornithine decarboxylase and cyclic AMP-dependent protein kinase activities.     

G1 specific increases in cyclic AMP levels and protein kinase activity in Chinese hamster ovary cells.

Chinese hamster ovary cells were synchronized by selective detachment of cells in mitosis. The adenosine 3':5'-cyclic monophosphate (cyclic AMP) intracellular concentrations and cyclic AMP-dependent protein kinase activities were measured as these cells traversed G1 phase and entered S phase. Protein kinase activity, assayed in the presence or absence of saturating exogenous cyclic AMP in the reaction mixture, was lowest in early G1 phase (2 h after mitosis), increased 2-fold (plus exogenous cyclic AMP in reaction mixture) or 3.5-fold (minus cyclic AMP in reaction mixture) to maximum values in mid to late G1 phase (4-5 h after mitosis), and then decreased as cells entered S phase. Intracellular cyclic AMP concentrations were minimal 1 h after mitosis, increased 5-fold to maximum levels at 4-6 after mitosis, and decreased as cells entered S phase. Similar to the fluctuations in intracellular cyclic AMP, the cyclic AMP-dependent protein kinase activity ratio increased more than 40% in late G1 or early S phase. Puromycin (either 10 mug/ml or 50 mug/ml) administered 1 h after mitosis inhibited cyclic AMP-dependent protein kinase activity up to 50% by 5 h after mitosis, while similar treatment (10 mug/ml) had no effect on the increase in cyclic AMP formation. These data demonstrate that: (1) total protein kinase activity changed during G1 phase and this increase was dependent on new protein synthesis; (2) the increased intracellular concentrations of cyclic AMP were not dependent on new protein synthesis; and (3) the activation of cyclic AMP-dependent protein kinase was temporally coordinated with increased intracellular concentration of cycli AMP as Chinese hamster ovary cells traversed G1 phase and entered S phase. These results suggest that cyclic AMP acts during G1 phase to regulate the activation of cyclic AMP-dependent protein kinase.     

The rapid activation of tyrosine hydroxylase by the subcutaneous injection of formaldehyde

Ten minutes following the subcutaneous injection of formaldehyde, there is a 2-fold activation of adrenal medulla tyrosine hydroxylase. Enzyme activation appears to involve a shift of a fraction of the low affinity form of the enzyme to a higher affinity form. In the nonstessed rat approximately 29% of adrenal TH is in the activated (low Km) form. Following formaldehyde injection, approximately 54% of the enzyme is in the activated (low Km) form. The addition of cyclic AMP, ATP and Mg⁺⁺, in the presence of endogenous cyclic AMP-dependent protein kinase, to the enzyme obtained from adrenals of either the stressed or nonstressed rats increased the activity of both enzyme preparations to the same level. These data indicate that acute stress activates adrenal tyrosine hydroxylase in the rat and that a cyclic AMP-dependent protein phosphorylating system mediates the activation.     

Effects of methyl xanthine derivatives on cyclic AMP levels and ornithine decarboxylase activity of rat tissues

The administration of aminophylline results in rapid increases in cyclic AMP in the adrenal medulla, adrenal cortex, liver, and kidney of the rat. The injection of theophylline results in a similar increase in cyclic AMP in the liver of the rat. In all instances, these increases are followed by 4- to 2-fold elevations of ornithine decarboxylase activity. The generality of this phenomena suggests that ornithine decarboxylase activity is regulated by an increase in cyclic AMP.     

Effects of Ca2+ ionophore A23187, 1,2-dioctanoylglycerol, and dibutyryl cAMP on the activity and expression of ornithine decarboxylase in guinea pig lymphocytes

The Ca2+ ionophore A23187 induced small increases in ornithine decarboxylase activity and ornithine decarboxylase mRNA in guinea pig lymphocytes. 1,2-Dioctanoylglycerol potentiated the A23187-induced ornithine decarboxylase activity and the accumulation of mRNA for this enzyme. Dibutyryl cAMP also potentiated the enzyme activity, but had little effect on the accumulation of mRNA. 1,2-Dioctanoylglycerol and 12-O-tetradecanoylphorbol-13-acetate potentiated ornithine decarboxylase activity that had been increased by treatment with both A23187 and dibutyryl cAMP with a consistent increase in the ornithine decarboxylase mRNA. However, dibutyryl cAMP augmented ornithine decarboxylase activity that had been increased by the combination of A23187 and 1,2-dioctanoylglycerol without affecting the ornithine decarboxylase mRNA level. These results suggest that the protein kinase C and cyclic AMP pathways are involved in the enhancement of ornithine decarboxylase activity in guinea pig lymphocytes, but that the mechanisms of the enhancement differ for each pathway, the former increasing the ornithine decarboxylase mRNA level, but not the latter.     

Effects of adrenalectomy on CRH regulation of ACTH release: adenylate cyclase activity, cyclic AMP-dependent protein kinase activity and ACTH release

Corticotropin releasing hormone (CRH) stimulation of ACTH release and cyclic AMP-mediated events involved in the control of ACTH release were compared in sham-operated and adrenalectomized rats. CRH-stimulated adenylate cyclase activity was decreased in pituitary homogenates from adrenalectomized animals. CRH-stimulated cyclic AMP accumulation was essentially abolished and CRH-stimulated cyclic AMP-dependent protein kinase (A-kinase) activity was decreased in freshly prepared anterior pituitary cells from adrenalectomized animals. Basal and CRH-stimulated ACTH release was elevated in these cells. Since ACTH release is increased in adrenalectomized rats despite the down regulation of CRH-linked pituitary mechanisms, we speculate that the site of action of disinhibition by corticosterone of ACTH release (or synthesis) following adrenalectomy is distal to the generation of cyclic AMP and/or that non-CRH mediated mechanisms assume a greater role in ACTH regulation following adrenalectomy.     

Comparison of cyclic nucleotide specificity of guanosine 3',5'-monophosphate-dependent protein kinase and adenosine 3',5'-monophosphate-dependent protein kinase.

Guanosine 3',5'-monophosphate-dependent protein kinase (cyclic GMP-dependent protein kinase) and adenosine 3',5'-monophosphate-dependent protein kinase (cyclic AMP-dependent protein kinase) exhibited a high degree of cyclic nucleotide specificity when hormone-sensitive triacylglycerol lipase, phosphorylase kinase, and cardiac troponin were used as substrates. The concentration of cyclic GMP required to activate half-maximally cyclic dependent protein kinase was 1000- to 100-fold less than that of cyclic AMP with these substrates. The opposite was true with cyclic AMP-dependent protein kinase where 1000- to 100-fold less cyclic AMP than cyclic GMP was required for half-maximal enzyme activation. This contrasts with the lower degree of cyclic nucleotide specificity of cyclic GMP-dependent protein kinase of 25-fold when histone H2b was used as a substrate for phosphorylation. Cyclic IMP resembled cyclic AMP in effectiveness in stimulating cyclic GMP-dependent protein kinase but was intermediate between cyclic AMP and cyclic GMP in stimulating cyclic AMP-dependent protein kinase. The effect of cyclic IMP on cyclic GMP-dependent protein kinase was confirmed in studies of autophosphorylation of cyclic GMP-dependent protein kinase where both cyclic AMP and cyclic IMP enhanced autophosphorylation. The high degree of cyclic nucleotide specificity observed suggests that cyclic AMP and cyclic GMP activate only their specific kinase and that crossover to the opposite kinase is unlikely to occur at reported cellular concentrations of cyclic nucleotides.     

Induction of ornithine decarboxylase activity by growth and differentiation factors in FRTL-5 cells

Induction of ornithine decarboxylase has been correlated with the onset of cellular proliferation and cAMP production. Whether the resulting increases in polyamine levels are essential mediators of growth and/or differentiation or are merely incidental remains controversial. We have used FRTL-5 thyroid cells in culture to study the effects of three growth factors on ornithine decarboxylase activity. These factors [TSH, bovine calf serum, and 12-O-tetradecanoylphorbol-13-acetate (TPA)] are thought to act through different intracellular pathways. TSH stimulates cAMP production in thyroid cells, calf serum acts through ill-defined pathways to stimulate growth, and TPA is known to activate protein kinase C. Bovine calf serum and TSH acted synergistically to induce ornithine decarboxylase activity. Activity was maximal when the phosphodiesterase inhibitor, methyl isobutyl xanthine, was included. Individually, neither serum nor TSH was a potent stimulator of the enzyme. Ornithine decarboxylase mRNA was apparent on Northern blots as a doublet following one hour of exposure to these agents. TPA did not stimulate ornithine decarboxylase activity and had an inhibitory effect on enzyme induction by TSH and serum. Difluoromethylornithine, a specific inhibitor of ornithine decarboxylase, inhibited growth induced by both TPA and TSH in putrescine-free medium. This effect was not apparent in medium containing 10(-5) M putrescine. The data indicate that, although intracellular levels of cyclic AMP regulate ornithine decarboxylase activity, a component in serum is necessary for significant induction of this enzyme. Factors stimulating growth by non-cyclic AMP-dependent pathways may act without apparently stimulating this enzyme, although polyamines appear to be essential for their growth stimulatory effects.     

Altered cyclic AMP-dependent protein kinase activity in a mutant adrenocortical tumor cell line

A somatic cell genetic approach has been used to evaluate the role of cyclic AMP-dependent protein kinase in ACTH action on adrenal steroidogenesis. A mutant clone, 8BrcAMP^r-1, previously was isolated from an ACTH-sensitive adrenocortical tumor cell line (clone Y1) following mutagenesis and selective growth in 8-bromoadenosine 3′, 5′-monophosphate. This study demonstrates that the 8BrcAMP⁴-1 cells have an altered cyclic AMP-dependent protein kinase. The protein kinase in the cytosol of the mutant characteristically requires, for half-maximal activity, concentrations of cyclic AMP 7-fold higher than those required by the enzyme in preparations from the parent. The cytosolic cyclic AMP-dependent protein kinases of Y1 and 8BrcAMP^r-1 cells chromatograph similarly on columns of DEAE-cellulose. From each cell line, a major peak of activity (≥ 70% of recovered activity), designated as Peak I, elutes with 0.04–0.06 M NaCl; a second peak of activity, designated as Peak II, elutes with 0.12–0.14 M NaCl. Protein kinase activity in the Peak I fraction of mutant cells has a decreased apparent affinity (4-fold) for cyclic AMP relative to the corresponding fraction of parental Y1 cells. The protein kinase activities present in Peak II fractions from Y1 and mutant cells are indistinguishable. The protein kinase mutant exhibits poor steroidogenic responses to added ACTH and cyclic AMP; and as shown previously does not display the growth arrest and morphological changes produced in Y1 by these agents. These results suggest that cyclic AMP-dependent protein kinase is important in the regulation of adrenal steroidogenesis, morphology and growth by ACTH.     

Comparison of cyclic nucleotide specificity of guanosine 3′,5′-monophosphate-dependent protein kinase and adenosine 3′,5′-monophosphate-dependent protein kinase

Guanosine 3′,5′-monophosphate-dependent protein kinase (cyclic GMP-dependent protein kinase) and adenosine 3′,5′-monophosphate-dependent protein kinase (cyclic AMP-dependent protein kinase) exhibited a high degree of cyclic nucleotide specificity when hormone-sensitive triacylglycerol lipase, phosphorylase kinase, and cardiac troponin were used as substrates. The concentration of cyclic GMP required to activate half-maximally cyclic dependent protein kinase was 1000- to 100-folds less than that of cylic AMP with these substrates. The opposite was true with cyclic AMP-dependent protein kinase where 1000- to 100-fold less cyclic GMP was required for half-maximal enzyme activation. This contrasts with the lower degree of cyclic nucleotide specificity of cyclic GMP-dependent protein kinase of 25-fold when histone H2b was used as a substrate for phosphorylation. Cyclic IMP resembled cyclic AMP in effectiveness in stimulating cyclic GMP-dependent protein kinase but was intermediate between cyclic AMP and cyclic GMP in stimulating cyclic. AMP-dependent protein kinase. The effect of cyclic IMP on cyclic GMP-dependent protein kinase was confirmed in studies of autophosphorylation of cyclic GMP-dependent protein kinase where both cyclic AMP and cyclic IMP enhanced autophophorylation. The high degree of cyclic nucleotide specificity observed suggests that cyclic AMP and cyclic GMP activate only their specific kinase and that crossover to the opposite kinase is unlikely to occur at reported cellular concentrations of cyclic nucleotides.     

Differential activation of type-I and type-II adenosine 3'':5''-cyclic monophosphate-dependent protein kinases in liver of glucagon-treated rats.

The protein-bound cyclic AMP and the activity of cytosolic protein kinases in the presence and absence of cyclic AMP were determined in rat liver up to 2h after injection of glucagon. On the basis of the different salt-sensitivities of the activated cyclic AMP-dependent proteinkinases I and II, an activation of protein kinase II restricted to the high cyclic AMP concentrations present in the first 30 min after hormone injection was found. Essentially the same result was obtained by chromatographic analysis on DEAE-cellulose of liver cytosol from untreated rats and from rats killed at 2 and 60 min after glucagon injection. Protein kinase II activation was only detected at 2 min after injection. In contrast, the cyclic AMP-dependent protein kinase I was found to be nearly totally activated at 2 min and to be still almost as active at 60 min after the hormone stimulus, whereas the amount of bound cyclic AMP and the activation of total cytosolic protein kinases had fallen to two-thirds of their maximal values during this time period. A third cyclic AMP-independent protein kinase, which co-chromatographed with protein kinase type II, could be clearly distinguished from the two cyclic AMP-dependent kinases by use of the heat-stable inhibitor from bovine muscle, which totally inhibited the cyclic AMP-dependent enzymes, but stimulated the cyclic AMP-independent protein kinase.     

Cyclic AMP-dependent protein kinase is not involved in the in vivo activation of tyrosine hydroxylase in the adrenal gland after decapitation

Tyrosine hydroxylase is activated in the adrenal gland in vivo after acute stresses, such as decapitation or electroconvulsive shock. In nonstressed animals that are anesthetized with pentobarbital prior to surgical removal of the adrenals, approximately 5-10% of the enzyme molecules are in the activated form, whereas in stressed animals, approximately 40-50% of the enzyme molecules are in the activated form. In the present study, we have tested the hypothesis that the stress-induced activation of the adrenal enzyme in vivo is due to the phosphorylation of the enzyme by cyclic AMP-dependent protein kinase. Soluble adrenal tyrosine hydroxylase prepared from either stressed or nonstressed rats is incubated in vitro with [gamma-32P]ATP and purified cyclic AMP-dependent protein kinase under optimal conditions for the phosphorylation of the enzyme. Using this assay, we have measured the number of vacant sites remaining on the enzyme, which are available for in vitro phosphorylation by cyclic AMP-dependent protein kinase. These studies suggest that the initial, in vitro rate of phosphorylation of tyrosine hydroxylase isolated from stressed rats is less than the initial rate of phosphorylation of the enzyme isolated from nonstressed rats. However, there is no significant difference in the final level of 32P phosphorylation of tyrosine hydroxylase isolated from either stressed or nonstressed rats. We conclude that, even though phosphorylation of tyrosine hydroxylase by cyclic AMP-dependent protein kinase leads to the activation of the enzyme under in vitro conditions, this mechanism cannot account for the activation of the enzyme in vivo in the adrenal gland following decapitation.     

Effect of thyroliberin on the concentration of adenosine 3'':5''-phosphate and on the activity of adenosine 3'':5''-phosphate-dependent protein kinase in prolactin-producing cells in culture.

1. The effects of thyroliberin were studied in cultured rat pituitary-tumour cells that synthesize and secrete prolactin (the GH4C1 cell strain). 2. Prolactin and cyclic AMP were measured by radioimmunological methods, and a cyclic AMP-dependent protein kinase was characterized by using histone as substrate. 3. Prolactin release was studied after 5-60min of treatment, and synthesis after 48h of treatment with thyroliberin. One-half maximum stimulation of release and synthesis were observed at 0.25 and at 4nM respectively. 4. Cyclic AMP was temporarily increased in cell suspensions after treatment with thyroliberin, and one-half maximum stimulation was observed at 25nM. 5. Dibutyryl cyclic AMP increased prolactin release and synthesis, one-half maximum effects being obtained at 20 micronM. 6. A cyclic AMP-dependent protein kinase, which was one-half maximally stimulated at 30 nM-cyclic AMP, was demonstrated. 7. An increase in the activity ratio (-cyclic AMP/+cyclic AMP) of the cyclic AMP-dependent protein kinase was observed after treatment with thyroliberin. Total protein kinase activity in the presence of cyclic AMP was unaltered. The time-course of enzyme activation was similar to that of cyclic AMP formation and corresponded to the time when prolactin release was first observed. 8. It is concluded that thyroliberin induces cyclic AMP formation, resulting in the activation of a cyclic AMP-dependent protein kinase.     

Growth regulatory effects of cyclic AMP and polyamine depletion are dissociable in cultured mouse lymphoma cells

Treatment of mouse lymphoma S49 cells with D,L-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, depleted cellular polyamine levels and stopped cell growth. The cells were arrested predominantly in G1. Thus, polyamine depletion may lead to a regulatory growth arrest in S49 cells. We tested two hypotheses regarding the relationship of growth arrest mediated by polyamine limitation to that mediated by cyclic AMP (cAMP). The hypothesis that cAMP-induced arrest results from polyamine depletion is not tenable, because the arrest could not be reversed by addition of exogenous polyamines, and because cellular polyamine levels do not drop in dibuturyl cyclic AMP (Bt2cAMP)-arrested cells. The hypothesis that polyamine-mediated growth arrest is effected via modulation of cAMP levels or cAMP-dependent protein kinase activity was also shown to be incorrect, because a S49 variant deficient in cAMP-dependent protein kinase was arrested by DFMO. The activities of the polyamine-synthesizing enzymes ornithine decarboxylase (ODC) and S-adenosyl methionine decarboxylase (SAMD) are both reduced in Bt2cAMP-treated cells to about 10% of that in control populations, as shown previously. DFMO diminishes ODC activity and augments SAMD activity in both untreated and Bt2cAMP-treated cells, leading to polyamine depletion in both cases.     

The role of protein kinase activation in the control of steroidogenesis by adrenocorticotrophic hormone in the adrenal cortex

A method has been developed for investigation of the effect of adrenocorticotrophic hormone (ACTH) on the state of activation of a cyclic AMP-dependent protein kinase within cells of the adrenal cortex. Enzyme activity was measured in terms of the quantity of (32)P transferred from [gamma-(32)P]ATP to histone under conditions in which bound cyclic AMP did not dissociate from the regulatory subunit of the protein kinase ACTH (1x10(-2)i.u./ml) caused a rapid and complete activation of the cyclic AMP-dependent protein kinase activity within 2min of hormone addition to the isolated cells. In response to a range of ACTH concentrations a sigmoid log dose-response curve for protein kinase activation was obtained, with half-maximal stimulation attained at about 1x10(-3)i.u./ml. However, some low doses of ACTH that elicited a marked (but submaximal) steroidogenic response failed to cause a clear stimulation of protein kinase activity in isolated adrenal cells. Theophylline (2mm) potentiated the effect of ACTH on protein kinase activity. The results implicate an important role for protein kinase in ACTH action on the adrenocortical cell.     

Hormonal control of liver regeneration

Two peaks in cyclic AMP production in rat livers 4 and 12h after partial hepatectomy (MacManus et al., 1972) were confirmed and a third peak established at 22h, which is the peak of DNA synthesis. The increases in cyclic AMP were prevented by beta-adrenergic blocking agents, propranolol and pindolol, without affecting ornithine decarboxylase induction or DNA synthesis. The alpha-blocking agents, phenoxybenzamine and phentolamine, given at the time of partial hepatectomy, delayed the rise in ornithine decarboxylase normally found 4h after operation, but did not affect DNA synthesis. If the alpha-blocking agents were given at 9-12h or 18h, the onset of DNA synthesis was delayed. Phenoxybenzamine did not affect the induction of ornithine decarboxylase in intact rat livers by glucagon or growth hormone, but did inhibit induction by dexamethasone. The induction of ornithine decarboxylase produced by dexamethasone was inhibited by 17alpha-hydroxy-progesterone; this compound also blocked the induction of ornithine decarboxylase in livers of partially hepatectomized rats.     

Changes in rat adrenal cyclic nucleotides during normal and neoplastic growth

In an attempt to correlate changes in cyclic nucleotide levels with in vivo growth of the rat adrenal gland we have measured adrenal cyclic AMP and cyclic GMP in normal, hyperplastic, and neoplastic rat adrenals. The first group of animals were subject to either unilateral adrenalectomy (ADX) or acute hypophysectomy 1 h prior to unilateral adrenalectomy (HADX). Cyclic nucleotides were measured in the contralateral adrenal post-operatively. In HADX rats cyclic GMP rose steadily throughout the 7 day study period, while ADX rats exhibited significant decreases in adrenal cyclic GMP. Cyclic AMP remained approximately 1.5 pm/mg tissue in HADX rats, while in ADX rats there was significant elevation of adrenal cyclic AMP at all time points. Cyclic GMP/cyclic AMP ratios remained constant in HADX animals; however, the growing adrenals of ADX animals exhibited depressed cyclic GMP/cyclic AMP ratios at all time periods.Adrenal hyperplasia was induced in a seond group of animals by a transplantable, corticotropin-secreting, pituitary tumor. Adrenals from age-matched animals served as controls. Adrenal cyclic AMP was significantly elevated in tumor-bearers at a time correspinding to the peak accumulation of adrenal weight, protein and DNA in these animals. In contrast, adrenal cyclic GMP in both tumor-beares and control animals fell steadily throughout the study period. Cyclic GMP/cyclic AMP ratios of control animals decreased from 2 to 3 weeks post-transplant remaining at the 3 week value during the period corresponding to rapid adrenal growth in tumor-bearers. The cyclic GMP/cyclic AMP ratio in the hyperplastic adrenals of tumor-bearers decreased steadily throughout their rapid growth period, suggesting a positive correlation between adrenal growth and depression of the cyclic GMP/cyclic AMP ratio.Cyclic nucleotide levels in neoplastic adrenals of rats bearing the transplantable adrenocortical carcinoma 494 were compared with cyclic nucleotides in normal rat adrenal glands. Cyclic AMP was not different in the two groups. However, the cyclic GMP content of neoplastic adrenals was significantly lower than that of normal adrenal tissue, causing a suppression of the cyclic GMP/cyclic AMP ratio in the neoplastic tissue. Thus, measurement of adrenal cyclic nucleotides in both hyperplastic and neoplastic rat adrenal glands suggests that adrenal growth in vivo may be characterized by a depression of the cyclic GMP/cyclic AMP ratio.