Forskolin Stimulates Adenylate Cyclase Activity, Cyclic AMP Accumulation, and Adrenocorticotropin Secretion from Mouse Anterior Pituitary Tumor Cells

Abstract: The effects of forskolin, an adenylate cyclase activator, were investigated on adrenocorticotropin (ACTH) secretion from AtT-20/D16-16 mouse pituitary tumor cells. Forskolin increased adenylate cyclase activity in these cells in the absence of added guanyl nu-cleotide, an effect blocked by somatostatin. Cyclic AMP synthesis and ACTH secretion increased in a concentration-dependent manner, not only in the clonal cells, but in primary cultures of rat anterior pituitary as well. Somatostatin inhibited cyclic AMP synthesis and ACTH secretion in response to forskolin. When forskolin was coapplied with corticotropin releasing factor, cyclic AMP synthesis was potentiated and ACTH secretion additive. The calcium channel blocker, nifedipine, inhibited forskolin, and 8-bromocyclic AMP stimulated ACTH secretion. These data suggest that ACTH secretion may be regulated at the molecular level by changes in cyclic AMP formation, which in turn regulate a calcium gating mechanism.     

Role of cyclic AMP and protein kinase on the steroidogenic action of ACTH, prostaglandin E1 and dibutyryl cyclic AMP in normal adrenal cells and adrenal tumor cells from humans.

The role of the cyclic AMP-protein kinase system in mediating the steroidogenic effect of ACTH, prostaglandin E1 and dibutyryl cyclic AMP, induced similar stimulations of protein kinase activity, cyclic AMP was studied using human adrenal cells isolated from normal and adrenocortical secreting tumors. At high concentrations of ACTH, complete activation of protein kinase of normal adrenal cells was observed within 3 min, at the time when cyclic AMP production was slightly increased and there was still no stimulation of steroidogenesis. At supramaximal concentrations, ACTH, PGE1 and dibutyryl cyclic AMP and cortisol productions in adrenal cells isolated from normal and from one adrenocortical tumor. In one tumor in which the adenylate cyclase activity was insensitive to ACTH, the hormone was unable to stimulate protein kinase or steroidogenesis, but the cells responded to both PGE1 and dibutyryl cyclic AMP. In another tumor in which the adenylate cyclase was insensitive to PGE1, this compound also did not increase protein kinase activity or steroidogenesis, but both parameters were stimulated by ACTH and dibutyryl cyclic AMP. After incubation of normal adrenal cells with increasing concentrations of ACTH (0.01-100 nM) marked differences were found between cyclic AMP formation and cortisol production. However at the lowest concentrations of ACTH exerting an effect on steroid production a close linked correlation was found between protein kinase activation and cortisol production, but half-maximal and maximal cortisol production occurs at lower concentration of ACTH than was necessary to induce the same stimulation of protein kinase. Similar findings were found after incubating the adrenal cells with dibutyryl cyclic AMP (0.01-10 mM). The results implicate an important role of the cyclic AMP-protein kinase system during activation of adrenal cell steroidogenesis by low concentrations of steroidogenic compounds.     

The stimulatory effect of corticotropin on cortisol biosynthetic pathway in guinea-pig adrenocortical cells

The mechanism of the prolonged stimulatory effect of corticotropin (ACTH) on adrenocortical synthesis of cortisol was studied in guinea-pig adrenocortical cells harvested from control animals and from guinea-pigs submitted 24 h before the sacrifice to a prolonged ether anesthesia in an attempt to induce a release of endogenous ACTH. As a result of this in vivo exposure to endogenous ACTH, the maximal capacity to produce glucocorticoids (by 1 X 10(5) cells incubated during 2 h) in response to ACTH increased from 579 +/- 111 ng (control group) to 915 +/- 143 ng for cells from treated animals, whereas the apparent affinity of the steroidogenic response to ACTH remained unchanged. This hyper-reactivity of cells from anesthetized animals was also evident in the presence of dibutyryl cyclic AMP. Moreover, there was increased conversion of exogenous pregnenolone into cortisol by cells from previously anesthetized animals. It was therefore concluded that ACTH increases in a lasting way the activity of steroidogenic pathway leading to cortisol synthesis by adrenocortical cells at sites distal to cyclic AMP generation. Besides an obvious increase of formation of pregnenolone in response to ACTH, it seems that this ACTH-induced enhancement in the capacity of the steroidogenic response to ACTH also implies a prolonged stimulatory influence of the peptide on the post-pregnenolone steroidogenic pathway leading to cortisol synthesis.     

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.     

Regulation of Guanosine Triphosphate Cyclohydrolase and Tetrahydrobiopterin Levels and the Role of the Cofactor in Tyrosine Hydroxylation in Primary Cultures of Adrenomedullary Chromaffin Cells

Selective modification of the tetrahydrobiopterin levels in cultured chromaffin cells were followed by changes in the rate of tyrosine hydroxylation. Addition of sepiapterin, an intermediate on the salvage pathway for tetrahydrobiopterin synthesis, rapidly increased intracellular levels of tetrahydrobiopterin and elevated the rate of tyrosine hydroxylation in the intact cell. Tyrosine hydroxylation was also enhanced when tetrahydrobiopterin was directly added to the incubation medium of intact cells. When the cultured chromaffin cells were treated for 72 h with N-acetylserotonin, an inhibitor of sepiapterin reductase, tetrahydrobiopterin content and the rate of tyrosine hydroxylation were decreased. Addition of sepiapterin or N-acetylserotonin had no consistent effect on total extractable tyrosine hydroxylase activity or on catecholamine content in the cultured chromaffin cells. Three-day treatment of chromaffin cell cultures with compounds that increase levels of cyclic AMP (forskolin, cholera toxin, theophylline, dibutyryl- and 8-bromo cyclic AMP) increased total extractable tyrosine hydroxylase activity and GTP-cyclohydrolase, the rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin. Tetrahydrobiopterin levels and intact cell tyrosine hydroxylation were markedly increased after 8-bromo cyclic AMP. The increase in GTP-cyclohydrolase and tetrahydrobiopterin induced by 8-bromo cyclic AMP was blocked by the protein synthesis inhibitor cycloheximide. Agents that deplete cellular catecholamines (reserpine, tetrabenazine, and brocresine) increased both total tyrosine hydroxylase and GTP-cyclohydrolase activities, although treating the cultures with reserpine or tetrabenazine resulted in no change in cellular levels of cyclic AMP. Brocresine and tetrabenazine increased tetrahydrobiopterin levels, but the addition of reserpine to the cultures decreased catecholamine and tetrahydrobiopterin content and resulted in a decreased rate of intact cell tyrosine hydroxylation in spite of the increased activity of the total extractable enzyme. These data indicate that in cultured chromaffin cells GTP-cyclohydrolase activity like tyrosine hydroxylase activity is regulated by both cyclic AMP-dependent and cyclic AMP-independent mechanisms and that the intracellular level of tetrahydrobiopterin is one of the many factors that control the rate of tyrosine hydroxylation.     

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.     

Dibutyryl cyclic AMP decreases glutamine synthetase in cultured 3T3-L1 adipocytes

Glutamine synthetase specific activity increases greater than 100-fold during the insulin-mediated differentiation of confluent 3T3-L1 cells into adipocytes. Incubation of the adipocytes for 22 h with 0.5 mM dibutyryl cyclic AMP plus 0.5 mM theophylline, 0.2 mM 8-bromo-cyclic AMP, 10 micro M epinephrine, or 1 microgram of alpha 1-24 adrenocorticotropic hormone/ml decreased glutamine synthetase by greater than 60%. During the same incubation period, there was no effect of these compounds on protein or on the specific activities of glucose-6-P dehydrogenase or hexokinase. In the presence of 0.5 mM theophylline, the dibutyryl cyclic AMP-mediated decrease in glutamine synthetase activity was half-maximal at 50 micro M dibutyryl cyclic AMP. Furthermore, between 10 micro M and 5 mM dibutyryl cyclic AMP, the dibutyryl cyclic AMP-mediated decrease in glutamine synthetase was similar in the absence or presence of 1 microgram of insulin/ml. Immunotitration of glutamine synthetase activity from 3T3 adipocytes indicates that the dibutyryl cyclic AMP-mediated decrease in the activity is due to a decrease in the cellular content of glutamine synthetase molecules. We studied the effects of dibutyryl cyclic AMP on the synthesis and degradation of glutamine synthetase. Synthesis rate was estimated from the incorporation of L-[35S]methionine into glutamine synthetase during a 60-min incubation period. Degradation rate was estimated from the first order disappearance of radioactivity from glutamine synthetase in 3T3 adipocytes previously incubated with L-[35S]methionine. Glutamine synthetase was isolated by immunoprecipitation followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Incubation of 3T3 adipocytes with dibutyrl cyclic AMP resulted in a rapid decline in the apparent synthesis rate of glutamine synthetase. In addition, dibutyryl cyclic AMP treatment increased the initial rate of glutamine synthetase degradation. The half-life of glutamine synthetase was 24.5 h in control cultures and 16 h in dibutyryl cyclic AMP-treated cultures. In contrast, dibutyryl cyclic AMP had little effect on the synthesis or degradation of soluble protein. Our data indicate that the dibutyryl cyclic AMP-mediated decrease in 3T3 adipocyte glutamine synthetase activity results from a decrease in the synthesis rate and an increase in the initial degradation rate of the enzyme.     

The influence of culture conditions on the induction of tyrosine aminotransferase by cyclic nucleotides in rat hepatoma cells.

The increase in tyrosine aminotransferase activity which occurs in rat hepatoma tissue culture (HTC) cells in response to cyclic AMP analogs has been shown to be an enzyme induction, similar to the larger response observed in certain other hepatoma cells and in liver. A specific antibody to tyrosine aminotransferase has been used to show that the number of enzyme molecules and the rate of enzyme synthesis are increased by N6,O2'-dibutyryl cyclic AMP in HTC cells. The effect on tyrosine aminotransferase is also produced by various 8-substituted derivatives of cyclic AMP and occurs whether or not the enzyme has been preinduced with a glucocorticoid. The response of the enzyme is greater when HTC cells are maintained in monolayer than in suspension cultures. Neither cell growth nor serum is required for the response.     

Coordinate regulation of adenylate cyclase, protein kinase, and specific enzyme synthesis by cholera toxin in hormonally unresponsive hepatoma cells

Since none of the hormones which activate adenylate cyclase in other tissues have been found to activate adenylate cyclase or to induce tyrosine aminotransferase in cultured Reuber hepatoma cells (H35), despite the stimulatory effects of cyclic AMP derivatives on the latter enzyme, we tested the ability of cholera toxin to influence these processes. At low concentrations cholera toxin was found to mimic the ability of cyclic AMP derivatives to selectively stimulate the synthesis of the aminotransferase. Adenylate cyclase and protein kinase activity were also enhanced, but only after a lag period as in other systems. Specific phosphorylation of endogenous H₁ histone was also shown to be increased by cholera toxin treatment. The increase in tyrosine aminotransferase activity is due to an increase in de novo synthesis as shown by radiolabeling experiments utilizing specific immunoprecipitation. The activity of another soluble enzyme induced by dibutyryl cyclic AMP, PEP carboxykinase, was also stimulated by exposure of H35 cells to cholera toxin. Combinations of cholera toxin and dexamethasone led to greater than additive increases in the activity of both the aminotransferase and carboxykinase. Close coupling of cyclic AMP production with protein kinase activation and enzyme induction was suggested by the observation that the ED₅₀ values for the stimulation of adenylate cyclase, cyclic AMP production, protein kinase, and tyrosine aminotransferase activities were found to be the same (5–7 ng/ml) within experimental error. The results indicate that the adenylate cyclase system in H35 cells is functionally responsive and they support the suggestion that activation of protein kinase is functionally linked to induction of specific enzymes.     

Glucocorticoid regulation of phenylethanolamine N-methyltransferase (PNMT) in organ culture of superior cervical ganglia

Glucocorticoid regulation of the adrenergic enzyme, phenylethanolamine N-methyltransferase (PNMT) was studied in organ cultures of the superior cervical ganglion (SCG) from newborn rats. Although PNMT catalytic activity was present in control ganglia, enzyme levels were too low to allow visualization of PNMT immunofluorescent cells. Addition of dexamethasone (DEX) or corticosterone to the medium resulted in a large increase in PNMT activity and bright PNMT immunoreactive (PNMT-IR) staining in cells resembling small, intensely fluorescent (SIF) cells. Addition of non-glucocorticoid steroids was ineffective. Exposure to a brief, 2-hr pulse of DEX (10(-6) M) in vitro elicited the same increase in PNMT as continual exposure to DEX. Studies using metabolic inhibitors demonstrated that the steroid-dependent increase in PNMT activity required both protein and RNA synthesis. Furthermore, the increase was inhibited by cytochalasin B and by the glucocorticoid receptor antagonists, DEX 21-mesylate and cortisol 21-mesylate. These observations suggest that glucocorticoids increase PNMT protein in SIF cells by interacting with specific steroid receptors that undergo translocation to the nucleus.     

Cyclic AMP-Dependent Induction of Serotonin N-Acetyltransferase Activity in Photoreceptor-Enriched Chick Retinal Cell Cultures: Characterization and Inhibition by Dopamine

The activity of serotonin N-acetyltransferase (NAT), a key regulatory enzyme in the melatonin biosynthetic pathway, was examined in low-density monolayer cultures of chick embryo retinal cells prepared with three levels of photoreceptor enrichment. In cultures prepared from embryonic day 8 retinas (E8), photoreceptors represented approximately 30% of the total cell population, whereas in those prepared from embryonic day 6 retinas (E6), approximately 70% of the cells were photoreceptors. In E8 retinas treated with kainic acid to destroy neurons (E8K), the relative content of photoreceptors was increased to approximately 50%. NAT activity was detectable in the cultures under all conditions studied, and was markedly increased by drugs that increase intracellular cyclic AMP levels and cyclic AMP-dependent protein kinase activity: 8-bromocyclic AMP, forskolin, and 3-isobutyl-1-methylxanthine (IBMX). Consistent with the hypothesis that NAT is localized in photoreceptors, the effects of the stimulatory treatments were significantly greater in E6 and E8K cultures than in E8 cultures. The stimulation of NAT activity in E6 cultures was inhibited by actinomycin D and cycloheximide, suggesting the involvement of RNA and protein synthesis. Dopamine inhibited the induction of NAT activity by forskolin and IBMX, but not that elicited by 8-bromocyclic AMP. The dopamine-mediated suppression of activity was significantly inhibited by pertussis toxin and by spiperone and sulpiride, both D2-dopamine receptor antagonists, but not by SCH 23390, a D1-dopamine receptor blocker, or antagonists of alpha-adrenergic, beta-adrenergic, or serotonergic receptors. Because the inhibitory effect of dopamine on E6 and E8K cultures was at least as great as that on E8 cultures, the results suggest that dopamine acts on D2-like receptors on photoreceptors. The receptors appear to be coupled to adenylate cyclase through an inhibitory GTP-binding protein and to mediate inhibition of cyclic AMP synthesis and consequent induction of NAT activity.     

Variations in some molecular events during the early phases of the Reuber H35 cell cycle. IV-regulation of tyrosine aminotransferase

Tyrosine aminotransferase activity increased during conversion of serum depleted quiescent Reuber H35 rat hepatoma cells into the proliferative state. Increased activity coincides with the actual increase of cells into S phase. The rate of tyrosine aminotransferase synthesis along the cell cycle was studied. The rate of enzyme synthesis fluctuated through the cell cycle but could not explain the increase of specific activity. Apparently enzyme activity is predominantly regulated by a post-translational event. Intracellular levels of cyclic AMP and cyclic GMP were measured at various times of G1 and S phases. In the early part of the cell cycle tyrosine aminotransferase decreased while intracellular levels of cyclic AMP increased. At later stages cyclic AMP rises concurrently with increased rates of enzyme synthesis. Induction of tyrosine aminotransferase by N6,O2'-dibutyryladenosine 3', 5'-monophosphate (Bt2cAMP) was studied. Inducibility by Bt2cAMP fluctuated through the cell cycle. Alternation of positive and negative control of tyrosine aminotransferase synthesis was observed. In early serum induced cells, Bt2cAMP increased enzyme activity without any increased rate of enzyme synthesis, on the contrary, a decreased rate of synthesis was observed. The data support the view that alternation of positive and negative control of tyrosine aminotransferase synthesis and temporary post-translational control of enzyme activity determine the enzyme level during the transition of quiescent hepatoma cells into proliferation.     

Pituitary cyclic AMP and plasma hormone responses to epinephrine administration in vivo

The present study was conducted to characterize the in vivo effects of epinephrine administration on levels of pituitary cyclic AMP and plasma hormones. Rats were injected with saline or epinephrine bitartrate (1 mg/kg lP) and sacrificed by decapitation 1, 5, 15, 30 or 60 min post-injection. Levels of pituitary cyclic AMP and plasma ACTH, beta-endorphin, beta-LPH, corticosterone and prolactin were determined by radioimmunoassays. The injection procedure itself was somewhat stressful as demonstrated by increased levels of plasma prolactin and ACTH 5 min following either saline or epinephrine injection. This "stress" response was rapid and short-lasting for the pituitary hormones. The response of the adrenal hormone, corticosterone, to saline injection was slower in onset and longer in duration. Pituitary cyclic AMP levels did not increase following saline injection. Epinephrine-injected animals displayed markedly elevated plasma levels of ACTH, beta-endorphin and beta-LPH at 15, 30 and 60 min as compared to control or saline-injected rats. In addition, levels of pituitary cyclic AMP were increased over 10 fold at these times. Levels of plasma prolactin, a stress-responsive hormone, were not significantly increased in epinephrine-injected animals as compared to saline-injected rats indicating that these later responses seem to be specific to epinephrine rather than to stress.     

The correlation of cyclic AMP and protein kinase activity in adipocytes with lipolysis stimulated by ACTH: the effect of adenosine deaminase and actinomycin D.

ACTH at levels as low as 0.05 mU/ml stimulated lipolysis, protein kinase and cyclic AMP accumulation in isolated fat cells from fed and fasted rats. Changes in cyclic AMP levels and in the protein kinase activity ratio were well correlated temporally. The protein kinase activity ratio was potentiated by adenosine deaminase. A sudden increase or decrease in either ACTH or dibutyryl cyclic AMP concentration was associated with a rapid and corresponding change in the rate of glycerol production. With ACTH, the changes in glycerol production were accompanied by appropriate changes in cyclic AMP levels. Actinomycin-D (10 UM) did not affect lipolysis or cyclic AMP accumulation activated by ACTH in fat cells.     

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.     

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.     

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.     

Fatty acids reverse the cyclic AMP inhibition of triacylglycerol and phosphatidylcholine synthesis in rat hepatocytes.

The influence of cyclic AMP analogues and fatty acids on glycerolipid biosynthesis in monolayer cultures of rat hepatocytes was investigated. Chlorophenylthio-cyclic AMP and adenosine 3':5'-cyclic phosphorothioate inhibited the rate of triacylglycerol synthesis from [1(3)-3H]glycerol, and phosphatidylcholine synthesis from [Me-3H]-choline. Supplementation of the hepatocytes with palmitate (1 mM) reversed chlorophenylthio-cyclic AMP inhibition of triacylglycerol synthesis. Similarly, cyclic AMP analogue-inhibition of phosphatidylcholine synthesis was abolished when the cells were simultaneously incubated with oleate (3 mM). Reactivation of phosphatidylcholine synthesis in chlorophenylthio-cyclic AMP-supplemented cells with oleate was accompanied by conversion of CTP: phosphocholine cytidylyltransferase into the membrane-bound form, since these cells released the enzyme more slowly after treatment with digitonin. The opposing actions of cyclic AMP and fatty acids are discussed in relation to the regulation of glycerolipid biosynthesis during starvation, diabetes and stress.     

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.