Effect of ascorbic acid on ACTH-induced cyclic AMP formation and steroidogenesis in isolated adrenal cells of vitamin E-deficient rats.

Isolated adrenal cells from Vitamin E-deficient and control rats were prepared by a trypsin digestion method. Cyclic adenosine 3',5'-monophosphate (cyclic AMP) formation was studied in response to adrenocorticotropin (ACTH) in the presence and absence of ascorbate by measuring the conversion of prelabeled adenosine 5'-triphosphate [14C]ATP to cyclic [14C]AMP. Ascorbate (0.5 mM) inhibited ACTH-induced cyclic [14C]AMP formation in adrenal cells isolated from Vitamin E-deficient rats but had no effect in the control cells. The inhibitory effect of ascorbate on ACTH-induced cyclic AMP formation in Vitamin E-deficient rats decreased as the concentration of ACTH increased. In Vitamin E-deficient rats ascorbate inhibited ACTH-induced cyclic [14C]AMP formation after 30 min of incubation. There was no further significant accumulation of cyclic [14C]AMP at 60 min or 120 min although in the absence of ascorbate cyclic [14C]AMP continued to be formed. The in vitro addition of alpha-tocopherol reduced the inhibition of ACTH-induced cyclic [14C]AMP formation by ascorbate in Vitamin E-deficient rats. These studies suggest that alpha-tocopherol and ascorbate may affect ACTH-induced cyclic AMP formation through interaction with the membrane-bound enzyme adenylate cyclase.     

Formation of adenosine 3′,5′-monophosphate from adenosine in mouse thymocytes

The effect of adenosine on the mouse thymocyte adenylate cyclase-adenosine 3′:5′-monophosphate (cyclic AMP) system was examined. Adenosine, like prostaglandin E₁, can cause 5-fold or greater increases in thymocyte cyclic AMP content in the presence but not in the absence of certain cyclic phosphodiesterase inhibitors. Two non-methylxanthine inhibitors potentiated the prostaglandin E₁ and adenosine responses, while methylxanthines selectively inhibited the adenosine response. Adenosine increased cyclic AMP content significantly wihtin 1 min and was maximal by 10 to 20 min with approx. 2 and 10 μM adenosine being minimal and half-maximal effective doses, respectively. Combinations of prostaglandin E₁, isoproterenol and adenosine were near additive and not synergistic. Of the adenosine analogues tested, only 2-chloro- and 2-fluoroadenosine significantly increased cyclic AMP. Thymocytes prelabeled with [¹⁴C] adenine exhibited dramatic increases in cyclic [¹⁴C]AMP 10 min after addition of adenosine or prostaglandin E₁ which corresponded to simultaneously determined increases in total cyclic AMP. Using [¹⁴C]adenosine, the percent of total cyclic AMP increase due to adenosine was only 16%. Adenosine was also shown to elicit a 40% increase in particulate thymocyte adenylate cyclase activity. Therefore, the increased content of cyclic AMP seen in mouse thymocytes after incubation with adenosine was due primarily to stimulation of adenylate cyclase and only partially to conversion of adenosine to cyclic AMP. The increased cellular content of cyclic AMP may be, in part, responsible for various immunosuppressive effects of adenosine.     

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.     

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.     

Stimulatory effect of gonadotropins on the synthesis of adenosine 3′ :5′-cyclic monophosphate and progesterone by suspensions of rat ovarian interstitial cells

A cell suspension was prepared from immature rat ovaries by treatment with trypsin and collagenase. The isolated cells were capable of converting [8-¹⁴C]adenine to cyclic [¹⁴C]AMP and the rate of this conversion was stimulated in vitro by luteinizing hormone and human chorionic gonadotropine, but not by prolactin, norepinephrine, dopamine or albumin. The accumulation of progesterone was also measured in these cells by radioimmunoassay. In vitro addition of luteinizing hormone stimulated the accumulation of radioimmunoassayable progesterone. The conversion of [8-¹⁴C]adenine to cyclic [¹⁴C]AMP showed a rapid increase during the first 30 min of the incubation period when luteinizing hormone was added to the incubation medium. Progesterone accumulation in response to the same dose of luteinizing hormone showed a lag period for the first 30 min of incubation after which there was an increase up to 2 h. The luteinizing hormone-induced progesterone accumulation was sensitive to puromycin, but there was no effect on the luteinizing hormone-induced increase in cyclic [¹⁴C]AMP formation from [8-¹⁴C]-adenine. Actinomycin D also inhibited the luteinizing hormone-induced progesterone accumulation, with no effect on cyclic AMP formation. The results suggest that the luteinizing hormone-induced progesterone accumulation in rat ovarian interstitial cell suspension is preceded by an increased accumulation of cyclic AMP and that the accumulation of steroid under the influence of luteinizing hormone involve processes sensitive to puromycin and antinomycin D.     

Regulation by lipolytic and antillipolytic compounds of the phosphorylation of specific proteins in isolated intact fat cells.

The effects of various lipolytic and antilipolytic compounds on the phosphorylation of specific proteins, on lipolysis, and on cyclic AMP levels have been studied in isolated intact fat cells of rats. Norepinephrine (NE), adrenocorticotropic hormone (ACTH), 3-isobutyl-1-methylxanthine (IBMX), and monobutyryl cyclic AMP (MBcAMP) each increased the incorporation of [³²P] into three proteins, with apparent molecular weights of approximately 130,000 (protein A), 69,000 (protein B), and 47,000 (protein C), as determined by gel electrophoresis in the presence of sodium dodecyl sulfate (DodSO₄^?). The concentrations of lipolytic agents necessary to obtain a half-maximal increase in phosphorylation of these proteins were similar to the concentrations necessary to obtain a half-maximal stimulation of lipolysis. Propranolol, a β-adrenergic blocking agent, blocked the effects of NE both on protein phosphorylation and on lipolysis, but did not modify the effects of ACTH, IBMX, or MBcAMP on these parameters. When the NE-induced increase in phosphorylation of proteins B and C was maximal, addition of propranolol resulted in a rapid dephosphorylation of these proteins and a rapid cessation of lipolysis; under the same experimental conditions, propranolol had almost no effect on the dephosphorylation of protein A. Concentrations of insulin that prevented or reversed the actions of NE and ACTH on lipolysis also prevented or reversed the NE- and ACTH-induced increase in [³²P] incorporation into proteins B and C. Insulin did not modify the effects of IBMX or MBcAMP either on lipolysis or on [³²P] incorporation into proteins B and C. Insulin increased the incorporation of [³²P] into a protein which, by several criteria, appeared to be protein A. Under a variety of experimental conditions in which lipolytic and antilipolytic hormones were studied, the rate of lipolysis correlated well with the level of phosphorylation of proteins B and C, but not with the level of cyclic AMP.     

Biosynthesis of steroids from cholesterol-14C by human adrenal carcinoma tissue

Slices prepared from human adrenal carcinoma tissue obtained froa a 51 year old female with virilism were incubated with cholesterol-4-¹⁴C (l) in the presence and absence of ACTH or cyclic AMP. The tissue homogenates were analyzed for steroids by the reverse isotope dilution method. Purification and identification of the radio-active metabolites were achieved by column, paper and thin-layer chromatography, derivative formation and crystallization to constant specific activity. Cholesterol-¹⁴C was converted to pregnenolone-¹⁴C (0.75%), progesterone- ¹⁴C (0.17%) and dehydroepiandrosterone-¹⁴C(0.15%). No evidence was found for the formation of labeled 17-hydroxyprogesterone, androstenedione, testosterone, and estrogens. Addition of ACTH to the incubations resulted in a two-fold inoreas of radioactive pregnenolone-¹⁴C and or dehydroepiandrosterone-¹⁴C. A two-fold increase of progesterone-¹⁴C synthesis was found in cyclic AMP-stimulated incubations.     

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.     

Cyclic AMP-induced release of [14C]taurine from pinealocytes.

Pinealocytes were prelabelled with [¹⁴C]taurine. Twenty-four hours later they were treated with derivatives of cyclic AMP. It was found that dibutyryl cyclic AMP and ?-chloro-phenyl-thio cyclic AMP treatment caused a large increase in the release of [¹⁴C]taurine. The effect of dibutryl cyclic AMP on [¹⁴C]taurine release was near maximal fifteen minutes after treatment started. In view of the known stimulatory effects of norepinephrine on pineal cyclic AMP and the recent discovery that norepinephrine causes the release of taurine from pinealocytes, one can conclude that norepinephrine stimulates [¹⁴C]taurine release from pinealocytes by acting through a cyclic AMP mechanism.     

Adrenocortical responses to adrenocorticotrophin in the rat

The time course of plasma adrenocorticotrophin (ACTH), adrenal cyclic AMP, adrenal corticosterone, and plasma corticosterone was measured in male Sprague-Dawley rats whose endogenous release of ACTH had been blocked (1) following rapid injections of 100 and 300 ng ACTH/100 g body weight, i.v., (2) during prolonged infusions at rates of 1, 2, and 4 ng ACTH/min per 100 g body weight, and (3) after termination of 30-min infusions at rates extending from 0.06 to 8 ng ACTH/min per 100 g body weight. Following injections, the time course of the variables is similar to the one simulated from our models of adrenal cortical secretion, including the simulation of an intermediate variable of our models of the adrenal cortex cell which was presumed to correspond to cyclic AMP. However, during prolonged infusions there is an unexpected overshoot of adrenal cyclic AMP content whereas adrenal and plasma corticosterone concentrations rise to a steady-state value without overshoot. The total amount of cyclic AMP gradually increases following the three increasing infusion rates of ACTH whereas similar levels of plasma corticosterone concentrations are reached at steady state; therefore the saturation of the adrenal cortical secretion is due to a step ulterior to cyclic AMP formation in the steroidogenesis. After 30-min infusions, plasma corticosterone concentration reaches its maximal value following a rate of ACTH input which evokes only a 4-fold increase in adrenal cyclic AMP content; however, there is a 250-fold increase of adrenal cyclic AMP with respect to control value following the higher rates of infusion of ACTH.     

Pituitary corticotropin-inhibiting peptide: Properties and use in study of corticotropin action

The biological properties of the naturally occurring pituitary peptide α_h^7–38-adrenocorticotropin (ACTH) have been investigated. α_h^7–38-ACTH is devoid of steroidogenic activity but inhibits competitively ACTH-induced steroidogenesis in vitro as well as in vivo. The long-term actions of ACTH on normal and tumor adrenal cells in culture are also antagonized by α_h^7–38-ACTH. The apparent K_i for the inhibition of cyclic AMP production by α_h^7–38-ACTH (301 ± 62 nm) was significantly higher than the apparent K_i for the inhibition of corticosterone synthesis (21.6 ± 6.8 nm). Analysis of the inhibition of ACTH-induced steroidogenesis and cyclic AMP production in normal rat adrenocortical cells indicates that two separate receptors may be involved in mediating these responses.     

Intracellular formation of analogues of cyclic AMP: Studies with brain slices labeled with radioactive derivatives of adenine and adenosine

A variety of radioactive analogs of adenine and adenosine were incubated with guinea pig cerebral cortical slices. Neither 1,N⁶-ethano[¹⁴C]adenosine nor 1,N⁶-ethanol[¹⁴C]adenine were significantly incorporated into intracellular nucleotides. 2-chloro[8-³H]adenine was incorporated, but at a very low rate and conclusive evidence for the formation of intracellular radioactive 2-chlorocyclic AMP was not obtained. N⁶-Benzyl[¹⁴C]adenosine was converted only to intracellular monophosphates and significant formation of radioactive N⁶-benzylcyclic AMP was not detected during a subsequent incubation. 2′-Deoxy-[8-¹⁴C] adenosine was converted to both intracellular radioactive 2′-deoxyadenine nucleotides and radioactive adenine nucleotides. Stimulation of these labeled slices with a variety of agents resulted in formation of both radioactive 2′-deoxycyclic AMP and cyclic AMP. Investigation of the effect of various other compounds on uptake of adenine or adenosine suggested that certain other adenosine analogs might serve as precursors of abnormal cyclic nucleotides in intact cells.     

Hormonal control of uterine contraction: Characterization of cyclic AMP-dependent membrane properties in the myometrium

A mitochondria-free membrane fraction prepared from rat myometrium accumulated ⁴⁵Ca²⁺ in the presence of oxalic acid and ATP. The rate of transport of Ca²⁺ into the membranous vesicles was increased by greater than 50% in the presence of 3′,5′-cyclic AMP, but not by 2′,3′-cyclic AMP or 5′-AMP. Membrane ATPase activity was stimulated by cyclic AMP in a manner similar to Ca²⁺-transport. ATPase activity was stimulated by Mg²⁺; slight additional stimulation was obtained in the presence of Na⁺ and K⁺ but not in the presence of Ca²⁺. Despite the cyclic AMP sensitivity of membrane ATPase activity, the absence of any effect of inhibitors of Ca²⁺-transport suggest it has little to do with Ca²⁺ accumulation by the membranes.Cyclic AMP-induced increase in Ca²⁺-transport and membrane ATPase activity was duplicated in vivo by incubating uteri in 10⁻⁴ M isoproterenol prior to membrane isolation. Isoproterenol has been previously shown to increase myometrial cyclic AMP levels, and changes in Ca²⁺-transport by cell membranes in relation to intracellular cyclic AMP levels may be the mechanism through which hormones modulate uterine contractility.     

Observations on the binding of adenosine 3′:5′-monophosphate to cell membrane fragments from ox cerebral cortex

Microsomal or synaptosome membrane fragments from ox brain bind cyclic AMP with a pH optimum of 7.0. Scatchard analysis shows the presence of at least two binding sites. Cyclic GMP and cyclic IMP only inhibit binding at concentrations 5000 times that of cyclic AMP and even higher concentration ratios of ATP and AMP have no effect. Membrane fragments saturated with cyclic [³H] AMP lost less than 7 % of bound nucleotide on incubation at 0°C for 45 min but lost 25 % in the same period in the presence of 10 μM non-radioactive cyclic AMP.     

On the mechanism of action of cholera toxin on isolated rat adrenocortical cells Comparison with the effects of adrenocorticotropin on steroidogenesis and cyclic AMP output

The effects of cholera toxin on isolated rat adrenocortical cells have been investigated. Both steroid and cyclic AMP output from adrenal cells were increased by the toxin in a dose dependent fashion. The concentration of toxin for half maximal stimulation for both of these responses was about 40 ng/ml. Maximal steroidogenesis and cyclic AMP output was obtained with similar concentrations of the toxin. A correlation was observed between the low amounts of cyclic AMP produced in response to all doses of cholera toxin and to physiologically significant concentrations of adrenocorticotropin (ACTH) (< 0.1 munit/ml; i.e. submaximal for steroidogenesis in this system). This was in direct contrast to the much higher levels of cyclic AMP generated by concentrations of ACTH greater than 1 munits/ml. Time course studies demonstrated a time-lag between toxin addition and steroid response of at least 40 min. Binding of cholera toxin to adrenal cells was rapid and was 90% complete within 15 min at both 37 and 0°C. These data indicate that most of the delay in response to cholera toxin is due to processes subsequent to the initial binding interaction. Following the initial delay the subsequent maximal rate of steroidogenesis brought about by cholera toxin was very similar to that obtained with a concentration of ACTH that was maximal for steroidogenesis. Significant increases in cyclic AMP levels were detected about 20 min before increased steroidogenesis was apparent. Possible explanations for this result are considered. The results presented indicate great potential use for cholera toxin in the study of adrenal steroidogenic control mechanisms, particularly at the level of receptor mechanisms and the role of cyclic AMP.     

An adenosine 3′,5′-monophosphate-requiring mutant of the luminous bacteria Beneckea harveyi

We have isolated a mutant of the luminous bacterium Beneckea harveyi, which requires exogenous adenosine 3′,5′-monphosphate (cyclic AMP) to snnthesize luciferase and emit light. The mutant was pleiotropic, lacking not only the ability to luminesce, but also the capacities to form flagella and the ability to utilize a variety of carbohydrates for growth. All these deficiencies could be corrected by added cyclic AMP. The cyclic AMP-induced de novo synthesis of luciferase was possible only ffter autoinduction had occurred. The induction time by cyclic AMP ranged between 6 and 10 min at 27°C.     

ATP increases chemoattractant induced cyclic GMP accumulation in Dictyostelium discoideum

Changes in guanosine cyclic 3′,5′-monophosphate associated with adenosine cyclic 3′,5′-monophosphate and folic acid addition in the presence of ATP have been examined in Dictyostelium discoideum. Preincubation with 1 mM ATP had no effect on the basal cyclic GMP level but increased the cycli GMP accumulation in response to cylci AMP (5·10⁻⁸ M) or folic acid (5·10⁻⁶ M) 40–50%. ATP could not be replaced by ADP of 5′-adenylyliminodiphosphate. Because ATP has no effect on cyclic AMP receptor binding these results indicate that structural membrane alterations (e.g. membrane phosphorylation) may control the transduction of a chemotactic signal.