Mechanism of corticotropin action on adrenal protein synthesis. The effects of inhibitors of protein synthesis and calcium chelators

We have recently shown that beside a general stimulation of most adrenal proteins, corticotropin induces a marked increase in a specific adrenal cytosolic protein, protein E, in intact and hypophysectomized rats. To further clarify the mechanisms by which corticotropin exerts its trophic action we have investigated the effects of cycloheximide, calcium and calcium chelator administration on intact and hypophysectomized animals. These substances were injected in rats with or without corticotropin, and slices of adrenal glands from control and treated animals were removed 5 h later, incubated with [14C]- or [3H]-leucine for 2 h, and cytosolic proteins analyzed by polyacrylamide gel electrophoresis using a dual labelling technique. When high doses of cycloheximide (higher than 500 micrograms) were injected in rats, incorporation of labelled leucine in adrenal slices of control and corticotropin-treated animals was inhibited. With 500 micrograms cycloheximide per rat, incorporation of labelled leucine in adrenal slices of control animals was normal, but the corticotropin stimulation of both protein E and total protein synthesis was inhibited. Lower doses of cycloheximide (100 micrograms per rat) completely inhibited the stimulatory effect of corticotropin on total protein synthesis but did not affect protein E synthesis, while after 50 micrograms per rat both stimulatory effects were preserved. The two higher doses of cycloheximide (500 and 100 micrograms per rat) could not completely block the steroidogenic effect of the hormone. The effects of calcium and calcium chelators were studied in 1-day hypophysectomized rats. Calcium alone or injected simultaneously with corticotropin has no effect. Calcium chelators injected simultaneously with corticotropin partially inhibited the stimulatory effects of corticotropin on steroidogenesis but totally inhibited stimulation of total protein synthesis, while the stimulation of protein E persisted. Our results show that after corticotropin, stimulation of protein E synthesis correlates better with steroidogenesis than with total protein synthesis.     

Regulation of the synthesis of lutropin-induced protein in rat testis Leydig cells

The mechanism of action of lutropin on the stimulation of the synthesis of a specific lutropin-induced protein in rat testis Leydig cells was investigated. Lutropin-induced protein has a mol.wt. of approx. 21000 and is detected by labelling the Leydig-cell proteins with [³⁵S]methionine, followed by separation by polyacrylamide-gel electrophoresis and radioautography of the dried gel. The incorporation of ³⁵S into lutropin-induced protein was used as an estimate for the synthesis of the protein. Incubation of Leydig cells with dibutyryl cyclic AMP or cholera toxin also resulted in the stimulation of synthesis of the protein. Synthesis of lutropin-induced protein, when maximally stimulated with 100ng of lutropin/ml, could not be stimulated further by addition of dibutyryl cyclic AMP. Addition of 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, further increased synthesis of the protein in the presence of a submaximal dose of lutropin (10ng/ml) but not in the absence of lutropin or with maximal amounts of lutropin (100 and 1000ng/ml). Actinomycin D prevented the effect of lutropin on the stimulation of lutropin-induced protein synthesis when added immediately or 1h after the start of the incubation, but not when added after 5–6h. This is interpreted as reflecting that, after induction of mRNA coding for lutropin-induced protein, lutropin had no influence on the synthesis of the protein in the presence of actinomycin D. Synthesis of the protein was also stimulated in vivo by injection of choriogonadotropin into rats 1 day after hypophysectomy, and the time course of this stimulation of lutropin-induced protein synthesis in vivo was similar to that obtained by incubating Leydig cells in vitro with lutropin. From these results it is concluded that stimulation of lutropin-induced protein synthesis by lutropin is most probably mediated by cyclic AMP and involves synthesis of mRNA.     

Effect of corticotropin treatment in vivo on the synthesis of a specific adrenal cytosolic protein. Characterization by dual-labelling technique and polyacrylamide-gel electrophoresis.

The effect of corticotropin in vivo on total and specific protein synthesis in the adrenal was studied. Adrenal slices from control and corticotropin-treated animals were incubated with [14C]- and [3H]-leucine respectively, followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of subcellular components. With this sensitive dual-labelling technique the following results were obtained. There was a general trophic effect on most adrenal proteins, but corticotropin produced a marked stimulation of a specific adrenal cytosolic protein. This protein has mol.wt. approx. 30 000 and pI 5.5. Corticotropin increased the incorporation of labelled leucine into proteins within 4 h, but no effect was observed before 2 h and after 16 h there was no further increase. These data suggest that this protein is not involved in the corticosteroidogenic action of corticotropin, but rather in the trophic action of this hormone.     

A MODULATING ROLE OF PITUITARY-ADRENAL AXIS IN CEREBRAL METABOLISM OF ADENOSINE 3'',5''-MONOPHOSPHATE

Abstract— The effect of adrenalectomy or hypophysectomy on the metabolism of adenosine 3',5'-monophosphate (cyclic AMP) in the cerebral cortex of male Wistar rats was investigated.
The bilateral removal of adrenal glands reduced significantly the activity of cerebral adenylate cyclase [EC 4.6.1.1]. whereas that of cyclic 3'.5'-nucleotide phosphodiesterase [EC 3.1.4.17] remained unchanged. The formation of cyclic AMP measured in cerebral cortical slices from adrenalectomized or hypophysectomized rats was also diminished. Decreases in the activity of adenylate cyclase and formation of cyclic AMP following adrenalectomy were antagonized by in vivo administration of dexamethasone or aldosterone, while those observed in hypophysectomized rats were restored by ACTH or dexamethasone. It is suggested that the pituitary adrenal axis has a modulating role in the metabolism of cerebral cyclic AMP, possibly by changing adenylate cyclase activity.     

Control of ovarian cholesterol ester biosynthesis

1. Experimental evidence is presented for a role of progesterone and 20alpha-hydroxypregn-4-en-3-one as inhibitors of cholesterol ester synthetase in the acute depletion of ovarian cholesterol ester after trophic stimulation. 2. Luteinizing hormone in vitro decreased by 84% the rate of esterification of cholesterol with added [(14)C]oleate by slices of rabbit ovarian interstitial tissue; this effect was mimicked by cyclic AMP (adenosine 3':5'-cyclic monophosphate) in vitro, and occurred without large changes in precursor pool sizes or membrane permeability. 3. Cyclic AMP was shown to have no direct effect on cholesterol ester synthetase or cholesterol esterase in cell-free extracts of rabbit ovarian interstitial tissue, but decreased the activity of cholesterol ester synthetase (not that of cholesterol esterase) in extracts prepared from slices previously incubated with it. 4. The inhibitory effect of cyclic AMP on esterification of cholesterol with added [(14)C]-oleate was prevented by both cycloheximide and aminoglutethimide phosphate (which also inhibited steroid synthesis in response to cyclic AMP). 5. Cyclic AMP raised the intracellular concentrations of progesterone and 20alpha-hydroxypregn-4-en-3-one in incubated slices by factors of 2.8 and 3.9 respectively. 6. Cycloheximide and aminoglutethimide phosphate administered in vivo blocked cholesterol ester depletion in response to luteinizing hormone in rats; in these ovaries cycloheximide and aminoglutethimide phosphate decreased the concentrations of progesterone and 20alpha-hydroxypregn-4-en-3-one and luteinizing hormone raised them. 7. Progesterone and 20alpha-hydroxypregn-4-en-3-one added to cell-free extracts of rabbit ovarian interstitial tissue in vitro (at concentrations comparable with those found in incubated slices) inhibited cholesterol ester synthetase by up to 85%. 8. The results are discussed with reference to the acute control of cholesterol ester concentrations in the ovary and adrenal cortex.     

Steroidogenesis in isolated adrenocortical cells. Correlation with receptor-bound adenosine 3′:5′-cyclic monophosphate

Because several groups have recently questioned a mediating role for cyclic AMP in adrenocortical steroidogenesis, we analysed the problem in more detail by measuring three different cyclic AMP pools in cells isolated from decapsulated rat adrenals. Extra-cellular, total intracellular and bound intracellular cyclic AMP were determined by radioimmunoassay in comparison with corticosterone production induced by low corticotropin concentrations. The increase in extracellular and total intracellular cyclic AMP with low corticotropin concentrations was dependent on the presence of a phosphodiesterase inhibitor and short incubation times. Bound intracellular cyclic AMP was less dependent on these two parameters. In unstimulated cells cyclic AMP bound to its receptor represents only a small fraction of the total intracellular cyclic AMP. After stimulation by a concentration of corticotropin around the threshold for corticosterone production, an increase in bound cyclic AMP was observed which correlated very well with steroidogenesis both temporally and with respect to corticotropin concentration. This finding was complemented by measuring a concomitant decrease in free receptor sites. Full occupancy of the receptors was not necessary for maximal steroidogenesis. Binding kinetics of cyclic [(3)H]AMP in concentrations equivalent to the intracellular cyclic AMP concentration suggest the presence of at least three different intracellular cyclic AMP pools. These observations are in agreement with a possible role for cyclic AMP as a mediator of acute steroidogenesis induced by low corticotropin concentrations.     

The effect of prostaglandins on ox pituitary content of adenosine 3′:5′-cyclic monophosphate and the release of growth hormone

1. An assay, based on competition between adenosine 3':5'-cyclic monophosphate (cyclic AMP) and cyclic [(3)H]AMP for binding to a rabbit skeletal muscle protein, has been used to measure tissue contents of cyclic AMP. The assay has a sensitivity of 0.05pmol of cyclic AMP. Cyclic GMP and cyclic CMP have 0.5%, and cyclic IMP 6.5%, of the ability of cyclic AMP to displace cyclic [(3)H]AMP from binding protein; AMP, ADP and ATP have no effect. 2. By using this method, the cyclic AMP content of ox pituitary slices exposed to prostaglandin was determined; release of growth hormone was measured by radioimmunoassay. 3. Release of growth hormone was increased by 45min incubation in 1mum-prostaglandin E(2) in the absence of theophylline, or in 10nm-prostaglandin E(2), 0.1mum-prostaglandin A(1) or 1mum-prostaglandin B(1) in the presence of 0.5mm-theophylline. 4. Pituitary cyclic AMP content was increased by 10min incubation in 1mum-prostaglandin E(2) in the absence of theophylline, or in 0.1mum-prostaglandin E(2) in the presence of 0.5mm-theophylline. 5. The maximum increase in cyclic AMP content was observed 10min, and significant changes in growth hormone release 30min, after introduction of prostaglandin E(2). 6. The increase in pituitary cyclic AMP content, but not in the rate of release of growth hormone, was observed in the absence of external Ca(2+). 7. The stimulation of release of growth hormone by prostaglandin was decreased by preincubation of tissue for 2h in colchicine (100mum) or cytochalasin B (10mug/ml). 8. These results support the suggestion that increased release of growth hormone after treatment with prostaglandin is the result of increased tissue cyclic AMP content, and possibly involves a microfilamentous or microtubular protein.     

Inhibition of steroidogenic response to corticotropin in mouse adrenal tumor cells (Y-1) by the ionophore A23187. Role of protein biosynthesis.

Addition of the ionophore A23187 to Y-1 mouse adrenal tumor cells in monolayer culture inhibits steroidogenesis and the steroidogenic response to corticotropin (50% inhibition at 1 . 10(-7)M). Inhibition is rapid in onset and is not overcome by addition of external Ca2+. The ionophore also inhibits stimulation of steroid synthesis by cyclic AMP. A23187 inhibits incorporation of the amino acid lysine into protein by Y-1 cells and the dose dependence of this inhibition closely resembles that of the inhibition of the steroidogenic response to corticotropin. Addition of A23187 to a subcellular system for protein synthesis prepared from Y-1 cells, inhibits incorporation of the amino acid phenylalanine into protein and this effect is not overcome by high concentrations of Ca2+. The inhibitory effect of A23187 on the response to corticotropin, like that response itself, takes place at some part of steroid synthesis after entry of cholesterol into the cells and before the side-chain cleavage of cholesterol. These studies confirm the importance of protein synthesis in the response to corticotropin and demonstrate that the effect of protein synthesized under the influence of corticotropin is exerted at some point in the events which bring substrate (cholesterol) to the mitochondrial side-chain cleavage enzyme system. It is also shown that A23187 inhibits protein synthesis, and hence the response to corticotropin, by a mechanism which is independent of the concentration of available Ca2+.     

Role of adenosine 3′:5′-cyclic monophosphate in the action of 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) on hepatic and renal metabolism

The possibility whether alterations in the cyclic AMP-adenylate cyclase-phosphodiesterase system play a role in the action of 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) on hepatic and renal carbohydrate metabolism was investigated. Administration of exogenous cyclic AMP (10mg/100g) was found to mimic the action of DDT which enhanced the activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase, fructose 1,6-diphosphatase and glucose 6-phosphatase in both liver and kidney cortex, elevated the concentration of blood glucose and urea and decreased the amount of hepatic glycogen. Treatment with theophylline augmented the effects of a submaximal dose of this halogenated hydrocarbon on serum urea and glucose as well as the key gluconeogenic enzymes in liver and kidney cortex. Addition of DDT in vitro to liver and kidney homogenates resulted in a significant enhancement of adenylate cyclase activity. Hepatic and renal slices from rats already treated with DDT displayed an increased ability to convert [(3)H]adenosine into cyclic [(3)H]AMP. Whereas kidney-cortex slices excised from rats given caffeine and DDT produced an even greater amount of cyclic [(3)H]AMP, imidazole, propranolol and hydrazine prevented the insecticide-stimulated rise in cyclic nucleotide production. In contrast, prostaglandin E(1) failed to exert any significant effect on DDT-induced increases in cyclic [(3)H]AMP synthesis from radioactive adenosine. The present study and our previous findings (Kacew & Singhal, 1973e) support the concept that the DDT-induced alterations in carbohydrate metabolism of liver and kidney cortex may be related to an initial stimulation of the cyclic AMP-adenylate cyclase system in these tissues.     

Hyperglucagonemia and altered responsiveness of hepatic adenylate cyclase-adenosine 3',5'-monophosphate system to hormonal stimulation during chronic ingestion of DL-ethionine.

Basal activity and hormonal responsiveness of the adenylate cyclase-adenosine 3',5'-monophosphate system were examined in premalignant liver from rats chronically fed the hepatic carcinogen DL-ethionine, and these data were correlated with endogenous levels of plasma glucagon. By 2 weeks basal hepatic cyclic AMP levels, determined in tissues quick-frozen in situ, were 2-fold higher in rats ingesting ethionine than in the pair-fed control. Enhanced tissue cyclic AM content was associated with an increase in the adenylate cyclase activity of whole homogenates of fresh liver from rats fed ethionine (68 +/- 5 pmol cyclic AMP/10 min per mg protein) compared to control (48 +/- 4). Cyclic AMP-dependent protein kinase activity ratios were also significantly higher (control, 0.38 +/- 0.04; ethionine 0.55 +/- 0.05) and the percent glycogen synthetase activity in the glucose 6-phosphate-independent form was markedly reduced (control, 52 +/- 7%; ethionine, 15 +/- 1.5%) in the livers of ethionine-fed rats compared to the controls, suggesting that the high total hepatic cyclic AMP which accompanied ethionine ingestion was bilogically effective. These changes persisted throughout the 38 weeks of drug ingestion. Immunoreactive glucagon levels, determined in portal venous plasma, were 8-fold higher than control after 2 weeks of the ethionine diet (control, 185 +/- 24 pg/ml; ethionine, 1532 +/- 195). Analogous to the changes in hepatic parameters, plasma glucagon levels remained elevated during the entire period of drug ingestion until the development of hepatomas. The hepatic cyclic AMP response to a maximal stimulatory dose of injected glucagon was blunted in vivo in ethionine-fed rats (control, 14 -fold increase over basal, to 8.63 +/- 1.1 pmol/mg wet weight; ethionine, 4.6-fold rise over basal, to 5.42 +/- 0.9). Reduced cyclic AMP responses to both maximal and submaximal glucagon stimulation were also evident in vitro in hepatic slices prepared from rats fed the drug, and the reduction was specific to glucagon. Absolute or relative hepatic cyclic AMP responses to maximally effective concentrations of protaglandin E1 or isoproterenol in hepatic slices from ethionine-fed rats were greater than or equal to those observed in control slices. Parallel alterations in hormonal responsiveness were observed in adenylate cyclase activity of whole homogenates of these livers, implying that the changes in cyclic AMP accumulation following hormone stimulation were related to an alteration in cyclic AMP generation in the premalignant tissue. In view of the recognized hepatic actions of glucagon and the desensitization of adenylate cyclase which can occur during sustained stimulation of the liver with this hormone, the endogenous hyperglucagonemia that accompanies ethionine ingestion could play a role in the pathogenesis of both the basal alterations in hepatic cyclic AMP metabolism and the reduced responsiveness to glucagon observed in liver from rats fed this carcinogen.     

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.     

Inbition of steroidogenic response to corticotropin in mouse adrenal tumor cells (Y-1 by the ionophore A23187 Role of protein biosynthesis

Addition of the ionophore A123187 to Y-1 mouse adrenal tumor cells in monolayer culture inhibits steroidogenesis and the steroidogenic response to corticotropin (50% inhibition at 1 · 10^?7 M). inhibition is rapid in onset and is not overcome by addition of external Ca²⁺. The ionophore also inhibits stimulation of steroid synthesis by cyclic AMP. A23187 inhibits incorporation of the amino acid lysine into protein by Y-1 cells and the dose dependence of this inhibition closely resembles that of the inhibition of the steroidogenic response to corticotropin. Addition of A23187 to a subcellular system for protein synthesis prepared from Y-1 cells, inhibits incorporation of the amino acid phenylalanine into protein and this effects and this effect is not overcome by high concentrations of Ca²⁺. The inhibitory effect of A23187 on the response to corticotropin, like that response itself, takes place at some part of steriod synthesis after entry of cholesteriol into the cells and before the side-chain cleavage of cholesterol. These studies confirm the importance of protein synthesis in the response to corticotropin and demonstrate that the effect of protein synthesized under the influence of corticotropin is exerted at some point in the events which bring substrate (cholesterol) to the mitochondrial side-chain cleavage enzyme system. It is also shown that A23187 inhibits protein synthesis, and hence the response to corticotropin, by a mechanism which is independent of the concentration of available Ca²⁺.     

Exposure of dog thyroid slices to acetylcholine induces refractoriness to its subsequent stimulation of glucose oxidation

An initial incubation of dog thyroid slices with 0.1 or 1 microM acetylcholine (ACH) for at least 2 h decreases its subsequent stimulation of [1-14C]glucose oxidation. Refractoriness persists for as long as 6 h in the absence of ACH. While new protein synthesis is essential for recovery, it is not necessary for its induction. Refractoriness is prevented when 25 microM tropicamide, an atropine-like drug, is present from the beginning of the initial incubation, but not when it is added after 2 h of incubation of slices with ACH, indicating that at this time ACH is no longer necessary for refractoriness. During refractoriness induced by ACH, stimulation of glucose oxidation by thyroid-stimulating hormone, prostaglandin E1, dibutyryl cyclic AMP, and cholera toxin, but not menadiol, is also significantly diminished. Incubation of thyroid slices with ACH does not modify its stimulation of iodide organification or 32Pi incorporation into phospholipids. These results suggest that the desensitization is not due to changes in the ACH receptor but rather to intracellular metabolic effects. This phenomenon may be important in the regulation of cholinergic effects on the thyroid.     

Is cyclic AMP the regulator of hepatic ornithine decarboxylase activity?

The role of cyclic AMP in the regulation of hepatic ornithine decarboxylase (ODC) activity in the rat was studied in the whole animal and in the perfused organ. Dibutyryl cyclic AMP or butyrate given to intact rats increased ODC activity; this increase was abolished by hypophysectomy 1 h prior to administering ether compound. Administration of 1 mg 1-methyl-3-isobutylxanthine (MIX) to intact rats increased ODC activity within 4 hours whereas hypophysectomy 1 h before treatment prevented this increase. No change in hepatic cyclic AMP content was seen in either intact or hypophysectomized rats following MIX. Perfusion with 0.5 mM dibutyryl cyclic AMP decreased ODC activity in isolated livers whereas perfusion with 0.5 mM 8-bromocyclic GMP produced a small increase in ODC activity. These data suggest that the effect of dibutyryl cyclic AMP in intact animals may be a property of the butyrate and that this action as well as the action of MIX may be mediated through the permissive effect of pituitary and/or adrenal hormones. The normal hepatocyte does not increase its ornithine decarboxylase activity after direct exposure to dibutyryl cyclic AMP.     

ACTH in vivo stimulation of the synthesis of a specific mitochondrial protein in the rat

Protein markers induced by hormones are the necessary probes to study hormone regulation of gene expression. We recently showed that ACTH was able to induce one of these markers in the cytosol of the rat adrenal (Dazord et al, Biochem. J. $\text{176}$, 233–239, 1978). In this paper we described another protein marker whose localization is mitochondrial and whose MW is 134 K. Maximal stimulation is seen 2 hours after ACTH injection. Actinomycin D injected 30 min before or 1 hour after the hormone blocks the stimulation. In hypophysectomized rats both ACTH and cyclic AMP are able to stimulate the synthesis of this protein.     

Metabolic regulation of steroidogenesis in isolated adrenal cells of rat. Relationship of adrenocorticotropin-, adenosine 3':5'-monophosphate-and guanosine 3':5'-monophosphate-stimulated steroidogenesis with the activation of protein kinase.

The data presented with the isolated adrenal cells, in the present study, show that adrenocorticotropin in the physiological concentration range stimulates the synthesis of guanosine 3':5'-monophosphate(cyclic GMP), protein kinase activity, and steroidogenesis in a concentration-dependent manner without detectable rise in the levels of adenosine 3':5'-monophosphate (cyclic AMP). Millimolar concentrations of cyclic AMP and cyclic GMP, which stimulate corticosterone synthesis, also activate kinase activity and steroidogenesis in a sigmoid concentration-response manner. The process of phosphorylation activated by corticotropin, cyclic AMP and cyclic GMP is not inhibited by cycloheximide or actinomyin D. It is therefore proposed that the hormonal responses mediated by cyclic GMP and cyclic AMP are via the protein kinase enzymatic steps, and the inhibitory effect of cycloheximide and actinomycin D in corticotropin-stimulated steroidogenesis follows this step. In conjuction with our previous observations that the biosynthetic steps from (20S)-20-hydroxycholesterol to corticosterone are neither inhibited by cycloheximide nor affected by cyclic GMP, it is inferred that the rate-limiting step of adrenal steroidogenesis is the transformation of cholesterol to (20S)-20hydroxycholesterol and this very step is regulated by cyclic GMP and cyclic AMP. Of further significance are the findings that micromolar cincentrations of cyclic AMP and cyclic GMP, which do not stimulate steroidogenesis, effectively stimulate protein kinase activity in a concentration-dependent manner. It is therefore concluded that all cyclic-nucleotide-dependent protein kinase activities of the cell are not necessarily related to steroidogenesis.     

Hyperglucagonemia and altered responsiveness of hepatic adenylate cyclase-adenosine 3′,3′-monophosphate system to hormonal stimulation during chronic ingestion of dl-ethionine

Basal activity and hormonal responsiveness of the adenylate cyclase-adenosine 3′,5′-monophosphate system were examined in premalignant liver from rat chronically fed the hepatic carcinogen DL-ethionine, and these data were correlated with endogenous levels of plasma glucagon. By 2 weeks basal hepatic cyclic AMP levels, determined in tissue quick-frozen in situ, were 2-fold higher in rats ingesting ethionine than in the pair-fed control. Enhanced tissue cyclic AMP content was associated with an increase in the adenylate cyclase activity of whole homogenates of fresh liver from rats fed ethionine (68 ± 5 pmol cyclic AMP/10 min per mg protein) compared to control (48 ± 4). Cyclic AMP-dependent protein kinase activity ratios were also significantly higher (control, 0.38 ± 0.04; ethionine 0.55 ± 0.05) and the percent glycogen synthetase activity in the glucose 6-phosphate-independent form was markedly reduced (control, 52 ± 7%; ethionine, 15 ± 1.5 %) in the livers of ethionine-fed rats compared to the controls, suggesting that the high total hepatic cyclic AMP which accompanied ethione ingestion was biologically effective. These changes persisted throughout the 38 weeks of drug ingestion. Immunoreactive glucagon levels, determined in portal venous plasma, were 8-fold higher than control after 2 weeks of the ethionine diet (contro, 185 ± 24 pg/ml; ethionine, 1532 ± 195). Analogous to the changes in hepatic parameters, plasma glucagon levels remained elevated during the entire period of drug ingestion until the development of hepatomas. The hepatic cyclic AMP response to a maximal stimulatory dose of injected glucagon was blunted in vivo in ethionine-fed rats (control, 14-fold increase over basal, to 8.63 ± 1.1 pmol/mg wet weight; ethionine, 4.6-fold rise over basal, to 5.42 ± 0.9). Reduced cyclic AMP responses to both maximal and submaximal glucagon stimulation were also evident in vitro in hepatic slices prepared from rats fed the drug, and the reduction was specific to glucagon. Absolute or relative hepatic cyclic AMP responses to maximally effective concentrations of prostaglandin E₁ or isoproterenol in hepatic slices from ethionine-fed rats were greater than or equal to those observed in control slices. Parallel alterations in hormonal responsiveness were observed in adenylate cyclase activity of whole homogenates of these livers, implying that the changes in cyclic AMP accumulation following hormone stimulation were related to an alteration in cyclic AMP generation in the premalignant tissue.In view of the recognized hepatic actions of glucagon and the desensitization of adenylate cyclase which can occur during sustained stimulation of the liver with this hormone, the endogenous hyperglucagonemia that accompanies ethionine ingestion could play a role in the pathogenesis of both the basal alterations in hepatic cyclic AMP metabolism and the reduced responsiveness to glucagon observed in liver from rats fed this carcinogen.     

Regulation by insulin of gluconeogenesis in isolated rat hepatocytes.

Insulin (10nM) completely suppressed the stimulation of gluconeogenesis from 2 mM lactate by low concentrations of glucagon (less than or equal to 0.1 nM) or cyclic AMP (less than or equal to 10 muM), but it had no effect on the basal rate of gluconeogenesis in hepatocyctes from fed rats. The effectiveness of insulin diminished as the concentration of these agonists increased, but insulin was able to suppress by 40% the stimulation by a maximally effective concentration of epinephrine (1 muM). The response to glucagon, epinephrine, or insulin was not dependent upon protein synthesis as cycloheximide did not alter their effects. Insulin also suppressed the stimulation by isoproterenol of cyclic GMP. These data are the first demonstration of insulin antagonism to the stimulation of gluconeogenesis by catecholamines. Insulin reduced cyclic AMP levels which had been elevated by low concentrations of glucagon or by 1 muM epinephrine. This supports the hypothesis that the action of insulin to inhibit gluconeogenesis is mediated by the lowering of cyclic AMP levels. However, evidence is presented which indicates that insulin is able to suppress the stimulation of gluconeogenesis by glucagon or epinephrine under conditions where either the agonists or insulin had no measurable effect on cyclic AMP levels. Insulin reduced the glucagon stimulation of gluconeogenesis whether or not extracellular Ca2+ were present, even though insulin only lowered cyclic AMP levels in their presence. Insulin also reduced the stimulation by epinephrine plus propranolol where no significant changes in cyclic AMP were observed without or with insulin. In addition, insulin suppressed gluconeogenesis in cells that had been preincubated with epinephrine for 20 min, even though the cyclic AMP levels had returned to near basal values and were unaffected by insulin. Thus insulin may not need to lower cyclic AMP levels in order to suppress gluconeogenesis.     

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.     

Polyoma virus and cyclic AMP-mediated control of dihydrofolate reductase mRNA abundance in methotrexate-resistant mouse fibroblasts.

As a model cell culture system for studying polyoma-mediated control of host gene expression, we isolated methotrexate-resistant 3T6 cells in which one of the virus-induced enzymes, dihydrofolate reductase, is a major cellular protein. In highly methotrexate-resistant cell lines dihydrofolate reductase synthesis accounts for over 10% that of soluble portein, corresponding to an increase of approximately 100-fold over the level in parental cells. This increase in dihydrofolate reductase synthesis is due to a corresponding increase in the abundance of dihydrofolate reductase mRNA and gene sequences. We have used these cells to show that infection with polyoma virus results in a 4- to 5-fold increase in the relative rate of dihydrofolate reductase synthesis and a corresponding increase in dihydrofolate reductase mRNA abundance. The increase in dihydrofolate reductase synthesis begins 15 to 20 h after infection and continues to increase until cell lysis. These observations represent the first direct evidence that viral infection of eukaryotic cells results in the increased synthesis of a specific cellular enzyme and an increase in the abundance of a specific cellular mRNA. In order to gain additional insight into the control of dihydrofolate reductase synthesis we examined other parameters affecting dihydrofolate reductase synthesis. We found that the addition of fresh serum to stationary phase cells results in a 2-fold stimulation of dihydrofolate reductase synthesis, beginning 10 to 12 h after serum addition. Serum stimulation of dihydrofolate reductase synthesis is completely inhibited by the presence of dibutyryl cyclic AMP as well as by theophylline or prostaglandin E1, compounds which cause an increase in intracellular cyclic AMP levels. In fact, the presence of dibutyryl cyclic AMP and theophylline results in a 2- to 3-fold decrease in the rate of dihydrofolate reductase synthesis and the abundance of dihydrofolate reductase mRNA. However, in contrast to the effect on serum stimulation, dibutyryl cyclic AMP and theophylline do not inhibit polyoma virus induction of dihydrofolate reductase synthesis or dihydrofolate reductase mRNA levels. These observations suggest that dihydrofolate reductase gene expression is controlled by at least two regulatory pathways: one involving serum that is blocked by high levels of cyclic AMP and another involving polyoma induction that is not inhibited by cyclic AMP.