Metabolic control mechanisms in mammalian systems. Involvement of adenosine 3':5'-cyclic monophosphate in androgen action.
ABSTRACT: 1. The ability of exogenously administered cyclic AMP (adenosine 3':5'-monophosphate) to exert andromimetic action on certain carbohydrate-metabolizing enzymes was investigated in the rat prostate gland and seminal vesicles. 2. Cyclic AMP, when injected concurrently with theophylline, produced marked increases in hexokinase, phosphofructokinase, glyceraldehyde phosphate dehydrogenase, pyruvate kinase, and two hexose monophosphate-shunt enzymes, as well as alpha-glycerophosphate dehydrogenase activity in accessory sexual tissues of castrated rats. The 6-N,2'-O-dibutyryl analogue of cyclic AMP caused increases of enzyme activity that were greater than those induced by the parent compound. 3. Time-course studies demonstrated that, whereas significant increases in the activities of most enzymes occurred within 4h after the injection of cyclic AMP, maximal increases were attained at 16-24h. 4. Increase in the activity of the various prostatic and vesicular enzymes was dependent on the dose of cyclic AMP; in most instances, 2.5mg of the cyclic nucleotide/rat was sufficient to elicit a statistically significant response. 5. Administration of cyclic AMP and theophylline also produced stimulation of enzyme activities in secondary sexual tissues of immature rats. 6. Cyclic AMP and theophylline did not affect significantly any of the enzymes studied in hepatic tissue. 7. Stimulation of various carbohydrate-metabolizing enzymes in the prostate gland and seminal vesicles by cyclic AMP was independent of adrenal function. 8. Concurrent treatment with actinomycin or cycloheximide prevented the cyclic AMP- and theophylline-induced increases in enzyme activities in both castrated and adrenalectomized-castrated animals. 9. Administration of a single dose of testosterone propionate (5.0mg/100g) to castrated rats caused a significant increase in cyclic AMP concentration in both accessory sexual tissues. 10. In addition, treatment with theophylline potentiated the effects of a submaximal dose of testosterone (1.0mg/100g) on all those prostatic and seminal-vesicular enzymes that are increased by exogenous cyclic AMP. 11. The evidence indicates that cyclic AMP may be involved in triggering the known metabolic actions of androgens on secondary sexual tissues of the rat.
Project description: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.
Project description:1. The hormonal regulation of phosphofructokinase was investigated in the accessory reproductive organs of the orchidectomized rat. 2. Phosphofructokinase activities declined to 51% and 47% in the prostate and 9% and 6% of the normal values in seminal vesicles 4 and 8 weeks after castration respectively. Administration of testosterone (100mug./100g. body wt.) for 3 days reversed substantially the effects of orchidectomy, and phosphofructokinase activity increased to 173% in the prostate and 536% in seminal vesicles as compared with the values of castrated controls. 3. Time-course studies demonstrated that after a single injection of testosterone (5mg./100g. body wt.) phosphofructokinase activity was maximally elevated to 236% in the prostate and 342% in seminal vesicles at 24hr. 4. Dose-response studies revealed that 2.5mg. of testosterone propionate/100g. body wt. was the minimal amount necessary to induce significant increases in enzyme activity in both accessory sex organs; maximal increases were obtained with a dose of 5mg./100g. body wt. 5. The observed enzyme increases induced by testosterone were inhibited by the simultaneous administration of oestradiol-17beta, and phosphofructokinase activity in this group of rats remained at 97% in the prostate and 137% of the control values in seminal vesicles. Oestradiol-17beta by itself failed to produce any significant effect on enzyme activity in either of these secondary sexual tissues. 6. The nature of the testosterone-induced increases in phosphofructokinase activity was studied by using a variety of inhibitors of RNA and protein synthesis. Cycloheximide, 5-fluorouracil and ethionine largely blocked the androgen-stimulated rise in enzyme activity observed 24hr. after steroid injection. The inhibitory effect of ethionine was completely reversed by the simultaneous administration of methionine. 7. Actinomycin, which is known to inhibit the synthesis of messenger RNA as well as the synthesis of other cellular RNA fractions, when given simultaneously with the hormone, also inhibited the testosterone-induced increases in prostatic and seminal-vesicular phosphofructokinase. However, when the antibiotic was given 6 or 12hr. after injection of the steroid, practically no inhibition of phosphofructokinase induction was obtained. This indicates that, once the enzyme-forming machinery is turned on and allowed to operate for a few hours, actinomycin is incapable of reversing the hormone-induced enzyme responses. 8. The results presented suggest that new RNA and protein synthesis may be involved in the observed androgen-induced increases in phosphofructokinase activity in the prostate and seminal vesicles of the orchidectomized rat.
Project description:Relationships between the cyclic AMP content, the rate of lipogenesis and the activity of acetyl-CoA carboxylase in acini prepared from lactating rat mammary tissue were investigated by exposing them to agents that increase their cyclic AMP content in the presence or absence of insulin. The dose-dependent inhibition of lipogenesis by theophylline in acini isolated from fed rats was highly correlated with the induced increases in acinar cyclic AMP content. Cyclic AMP of acini from 24 h-starved lactating rats was more sensitive in its response to theophylline than that in acini from fed animals. Neither forskolin nor a mixture of isoprenaline and Ro 7-2956 were able significantly to change either the rate of lipogenesis or the activity of acetyl-CoA carboxylase in acini from fed rats when added to incubations in vitro, in spite of the large increases in cyclic AMP concentration produced by these agents. Insulin was without effect on the activity of acetyl-CoA carboxylase and on either the basal or isoprenaline-stimulated cyclic AMP content of acini. These results are discussed in terms of the possibility that the rate of lipogenesis and the cyclic AMP content in mammary acini can vary independently of one another and of the activity of acetyl-CoA carboxylase.
Project description: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.
Project description:1. A comparison was made of the binding of 5alpha-dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) and cyclic AMP in the rat prostate gland. Distinct binding mechanisms exist for these compounds, and cyclic AMP cannot serve as a competitor for the 5alpha-dihydrotestosterone-binding sites and vice versa. In contrast with the results obtained with 5alpha-dihydrotestosterone, very small amounts of cyclic AMP are retained in nuclear chromatin and the overall binding of this cyclic nucleotide is not markedly affected by castration. 2. Androgenic stimulation does not lead to major increases in the adenylate cyclase activities associated with any subcellular fraction of the prostate gland. Accordingly, changes in the concentration of cyclic AMP in the prostate gland after hormonal treatment are likely to be small, but these were not measured directly. 3. When administered to whole animals in vivo, small amounts of non-degraded cyclic AMP are found in the prostate gland but sufficient to promote an activation of certain carbohydrate-metabolizing enzymes in the cell supernatant fraction. The stimulatory effects of cyclic AMP were not evident with cytoplasmic enzymes engaged in polyamine synthesis or nuclear RNA polymerases. These latter enzymes were stimulated solely by the administration of testosterone. 4. By making use of antiandrogens, a distinction can be drawn between the biochemical responses attributable to the binding of 5alpha-dihydrotestosterone but not of cyclic AMP. Evidence is presented to suggest that the stimulation of RNA polymerase, ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase is a consequence of the selective binding of 5alpha-dihydrotestosterone. Only the stimulation of glucose 6-phosphate dehydrogenase can be attributed to cyclic AMP or other metabolites of testosterone. 5. Overall, this study indicates that the formation of cyclic AMP is not a major feature of the androgenic response and affects only a restricted number of biochemical processes. Certainly, cyclic AMP cannot be considered as interchangeable with testosterone and its metabolites in the control of the function of the prostate gland. This difference is additionally emphasized by the failure of cyclic AMP to restore the morphology of the prostate gland in castrated animals; morphological restoration only follows the administration of androgens.
Project description:1. Concentrations of cyclic AMP (adenosine 3':5'-cyclic monophosphate) and rates of insulin release were measured in islets of Langerhans isolated from rat pancreas and incubated for various times in the presence of glucose, 3-isobutyl-1-methylxanthine, caffeine, theophylline, adrenaline and diazoxide. 2. Caffeine and theophylline produced small but significant increases in both cyclic AMP and release of insulin when they were incubated in the presence of 10mm-glucose. 3. 3-Isobutyl-1-methylxanthine produced a marked increase in the intracellular concentration of cyclic AMP in the presence of 5mm- and 10mm-glucose. However, insulin release was stimulated only in the presence of 10mm-glucose. 4. In response to rising concentrations of extracellular glucose (5-20mm) there was no detectable increase in the intracellular concentration of cyclic AMP even though there was a marked increase in the rate of insulin release. 5. In response to 10mm-glucose insulin release occurred in two phases and 3-isobutyl-1-methylxanthine potentiated the effect of glucose on both phases. The intracellular concentration of cyclic AMP remained constant with glucose and rose within 10min to its maximum value with 3-isobutyl-1-methylxanthine. 6. Adrenaline and diazoxide inhibited insulin release and lowered the intracellular concentration of cyclic AMP when islets were incubated with glucose or 3-isobutyl-1-methylxanthine. 7. It is suggested that glucose does not stimulate insulin release by increasing the concentration of cyclic AMP in islet cells. However, the concentration of cyclic AMP in islet cells may modulate the effect of glucose on the release process.
Project description:1. Human platelet nucleotides were labelled by incubating platelet-rich plasma with [U-(14)C]adenine. With such platelets, the effects of prostaglandin E1, theophylline and aspirin were determined on collagen-induced platelet aggregation and release of platelet ATP and ADP. Intracellular changes of platelet radioactive nucleotides, particularly 3':5'-cyclic AMP, were also determined both with and without collagen treatment. 2. Prostaglandin E1, theophylline and aspirin inhibited collagen-induced aggregation of platelets in a dose-dependent manner. Collagen-induced release of ATP and ADP and breakdown of radioactive ATP were also inhibited in a dose-dependent manner. 3. Prostaglandin E1 stimulated the formation of platelet radioactive 3':5'-cyclic AMP in a dose-dependent manner. With a given dose of prostaglandin E1, maximum formation of radioactive 3':5'-cyclic AMP occurred by 10-30s and thereafter the concentrations declined. The degree of inhibition of aggregation produced by prostaglandin E1, however, increased with its time of incubation in platelet-rich plasma before addition of collagen, so that there was an inverse relationship between the radioactive 3':5'-cyclic AMP concentration measured at the time of collagen addition and the subsequent degree of inhibition of aggregation obtained. 4. Neither theophylline nor aspirin at a concentration in platelet-rich plasma of 1.7mm altered platelet radioactive 3':5'-cyclic AMP contents. In the presence of prostaglandin E1, theophylline increased the concentration of radioactive 3':5'-cyclic AMP over that noted with prostaglandin E1 alone, but aspirin did not. 5. Mixtures of prostaglandin E1 and theophylline had a synergistic effect on inhibition of platelet aggregation. The same was true to a lesser extent with mixtures of prostaglandin E1 and aspirin. Such mixtures also inhibited collagen-induced release of platelet ATP and ADP and breakdown of platelet radioactive ATP. 6. Certain concentrations of either theophylline or aspirin and mixtures of small concentrations of prostaglandin E1 with either theophylline or aspirin caused little or no increase of radioactive 3':5'-cyclic AMP at the time of collagen addition, but inhibited aggregation to a marked degree, whereas higher concentrations of prostaglandin E1 alone caused a much greater increase of radioactive 3':5'-cyclic AMP at the time of collagen addition but inhibited aggregation to a lesser extent. With these compounds there does not appear to be a correlation between these parameters.
Project description:1. Adenosine was determined in rapidly frozen rat and guinea-pig brain and in guinea-pig cerebral tissues after incubation in vitro. Adenosine concentrations were approx. 2nmol/g wet wt. in frozen tissue, diminished at room temperature, and returned to 2nmol/g on incubation in oxygenated glucose/salines. 2. Superfusion with noradrenaline then increased the tissue's adenosine concentration 2.5-fold, and hypoxia caused an 8-fold increase. 3. Electrical stimulation alone or in the presence of noradrenaline or histamine increased the tissue's adenosine and cyclic AMP, but adenosine concentrations reached their peak later and were maintained for longer than those of cyclic AMP. 4. Superfusion with l-glutamate with and without electrical excitation raised adenosine concentrations to 15-34nmol/g. The increases in cyclic AMP on electrical stimulation, superfusion with glutamate or a combination of these treatments were diminished by addition of adenosine deaminase or theophylline. 5. It is concluded that adenosine can be produced endogenously in cerebral systems, in sufficient concentrations to accelerate an adenosine-activated adenylate cyclase, and by this route can contribute to the cerebral actions of electrical stimulation and of the neurohumoral agents. In certain instances cyclic AMP as substrate contributes to an increase in adenosine.
Project description:The influence of cyclic AMP on cartilage degradation was investigated by using phosphodiesterase inhibitors [theophylline and 3-isobutyl-1-methylxanthine (IBMX)], forskolin (which activates the catalytic subunit of adenylate cyclase) and cyclic AMP analogues (dibutyryl and 8-bromo). Breakdown was assessed by quantification of proteoglycans released into the media of 8-day bovine nasal-septum cartilage cultures. Theophylline (1-20 mM), IBMX (0.01-2 mM) and dibutyryl cyclic AMP (0.1-2 mM) had little or no influence on the rate of proteoglycan release from unstimulated (no-endotoxin) cartilages. A small but detectable increase in breakdown was observed with 8-bromo cyclic AMP (0.5-2 mM) and forskolin (50-75 micrograms/ml). To examine potential inhibitory influences of these agents, the cyclic AMP modulators were added to cultures simultaneously treated with Salmonella typhosa endotoxin (12-25 micrograms/ml), a potent stimulator of cartilage degradation. The 3-4-fold stimulation of breakdown by endotoxin was strikingly inhibited by all three classes of cyclic AMP regulators. Optimal inhibition was found at 10-20 mM-theophylline, 1-2 mM-IBMX, 50-75 micrograms of forskolin/ml, 2 mM-dibutyryl cyclic AMP and 2 mM-8-bromo cyclic AMP. Inhibition was shown to be reversible, indicating that cartilages were viable after treatment. Sepharose CL-2B chromatography of proteoglycan products released from treated cartilages showed that the endotoxin-stimulated shift to lower average Mr was significantly prevented by cyclic AMP analogues and phosphodiesterase inhibitors. Together, these results show that agents which increase cyclic AMP inhibit both quantitative and qualitative aspects of endotoxin-mediated cartilage degradation.
Project description:1. The basal and fluoride-stimulated activities of adenylate cyclase, and the maximal activities of 3':5'-cyclic AMP phosphodiesterase and 3':5'-cyclic GMP phosphodiesterase, together with the Km values for their respective substrates, were measured in muscle, liver and nervous tissues from a large range of animals to provide information on the mechanism of control of cyclic AMP concentrations in these tissues. High activities of adenylate cyclase and cyclic AMP diesterase are found in nervous tissues and in the more aerobic muscles (e.g. insect flight muscles, cardiac muscle and some vertebrate skeletal muscles). The activities of these enzymes in liver are similar to those in the heart of the same animal. The Km values for the enzymes from different tissues and animals are remarkably similar. 2. The comparison of cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase activities suggests that in vertebrate tissues only one enzyme (the high-Km enzyme), which possesses dual specificity, exists, whereas in invertebrate tissues there are at least two phosphodiesterases with separate specificities. 3. A simple quantitative model to explain the control of the steady-state concentrations of cyclic AMP is proposed. The maximum increase in cyclic AMP concentration predicted by comparison of basal with fluoride-stimulated activities of adenylate cyclase is compared with the maximum increases in concentration produced in the intact tissue by hormonal stimulation: reasonable agreement is obtained. The model is also used to predict the actual concentrations and the rates of turnover of cyclic AMP in different tissues and, where possible, these values are compared with reported values. Reasonable agreement is found between predicted and reported values. The possible physiological significances of different rates of turnover of cyclic AMP and the different ratios of high- and low-Km phosphodiesterases in different tissues are discussed.