Regulation of cyclic AMP level and lactic acid production in Ehrlich ascites tumor cells.

1. Quercetin (3.3',4',5,7-pentahydroxy flavone) at the concentration of 10(-4) M, as well as 2-10(-2) M theophylline and 1.5 - 10(-4) M prostaglandin E2 caused maximal rise of cyclic AMP in Ehrlich ascites tumor cells. 2. No additional increase of cyclic AMP level in these cells was found when both quercetin (10(-4) M) and theophylline (2-10(-2) M) were present in the incubation medium, while combination of quercetin (10(-4) M) and prostaglandin E2 (1.5 - 10(-4) M) has a synergistic effect on the level of cyclic AMP. 3. Degradation of cyclic AMP by homogenate of Ehrlich ascites tumor cells was inhibited by both quercetin and theophylline. 4. Quercetin, and to a smaller but significant extent theophylline, inhibited the lactic acid production in Ehrlich ascites tumor cells while prostaglandin E2 did not change the glycolytic rate in these cells. No synergistic inhibitory effect on lactic acid production was found when combinations of quercetin and prostaglandin E2, quercetin and theophylline or prostaglandin E2 and theophylline were tested. 5. Treatment of Ehrlich ascites tumor cells with dextran sulfate abolished the inhibitory effect of quercetin on lactic acid production, while the effect of the bioflavonoid on cyclic AMP levels was not altered.     

Involvement of cyclic AMP in the production of the acid protease Acp1 by Sclerotinia sclerotiorum

The acid protease Acp1 is produced by Sclerotinia sclerotiorum during plant infection. We explored the mechanism involved in the triggering of that production and found that cyclic AMP played a positive role. Acp1 could be produced in the sole presence of exogenous cyclic AMP. The use of molecules known to increase or decrease the intracellular cyclic AMP levels confirmed the impact of this nucleotide on the protease production and suggested its endogenous site of action. Further pharmacological studies showed the specific effect of cyclic AMP on Acp1 production and suggested that protein kinase A would be its likely target. Together, these results provide the first indication that the production of a pathogenesis-related fungal protease could depend on a cyclic AMP/Protein kinase A signalling pathway.     

Control of nucleotide and erythroascorbic acid pools by cyclic AMP in Neurospora crassa

UDPglucuronic acid and erythroascorbic acid were identified in extracts of the fungus Neurospora crassa. The concentrations of these two compounds are estimated, in growing wild type N. crassa, to be about 0.10 and 0.28 mumol/ml of cell water, respectively. The pools of these two compounds are regulated by cyclic AMP in Neurospora, both being elevated in the cr-1, adenylate cyclase deficient mutant and both being lowered by exogenous cyclic AMP. The pools of these two compounds are also elevated on nitrogen deprivation. The pools of a large number of other nucleotides are not influenced by cyclic AMP. Possible relationships between the metabolism of UDPglucuronic acid and erythroascorbic acid are discussed. It was found that exogenous cyclic AMP was much more effective in influencing cultures grown at 30-37 degrees C than those grown at 25 degrees C. We suggest that higher temperatures may render Neurospora more permeable to a variety of different compounds.     

Fatty acid control of cyclic AMP levels inNeurospora crassa

Several saturated, monosaturated, and polyunsaturated fatty acids produce rapid increases in cyclic AMP levels in the fungusNeurospora crassa when added to the growth medium at 10–50 M. The time courses of cyclic AMP increase resembled those previously shown to be induced by other agents, reaching peak cyclic AMP levels at about 2 min after fatty acid addition. These fatty acids had little or no influence on adenylate cyclase fromNeurospora crassa in vitro. On the basis of previous evidence that uncouplers of oxidative phosphorylation increase cyclic AMP levels and that fatty acids can act as uncouplers, we suggest that the fatty acids in vivo may act to increase cyclic AMP levels by acting as uncouplers of oxidative phosphorylation. In agreement with this suggestion, two fatty acids were shown to produce decreased ATP-ADP ratios inNeurospora at concentrations producing cyclic AMP increases.     

An insulin mediator preparation serves to stimulate the cyclic GMP activated cyclic AMP phosphodiesterase rather than other purified insulin-activated cyclic AMP phosphodiesterases

An insulin mediator preparation was obtained from rat hepatocytes which had been treated with insulin. This preparation inhibited adenylate cyclase activity. It stimulated the activity of homogeneous preparations of both the cytosolic and membrane-bound forms of rat liver cyclic GMP-activated cyclic AMP phosphodiesterase. It failed to activate homogeneous preparations of both the peripheral plasma membrane and 'dense-vesicle' cyclic AMP phosphodiesterases. The insulin mediator preparation stimulated cyclic GMP-activated cyclic AMP phosphodiesterase activity in a dose-dependent fashion with a hill coefficient of 0.46. Insulin caused the dose-dependent production of mediator activity in intact hepatocytes with a Ka of 9 pM, although concentrations of insulin greater than 10 nM progressively reduced stimulatory activity.     

Nicotinic acid reverses fasting ketosis by lowering the level of cyclic AMP.

When nicotinic acid was administered intraperitoneally to fasted rats, it reversed ketosis, decreased concentrations of cyclic AMP in adipose tissue and liver, and partially suppressed lipolysis. Administration of dibutyryl cyclic AMP reinduced ketosis in fasted rats previously treated with nicotinic acid. The results that nicotinic acid reverses ketosis by lowering tissue levels of cyclic AMP with a consequent suppression of lipolysis and ketogenesis.     

Forskolin potentiates adrenocorticotropin-induced cyclic AMP production and steroidogenesis in isolated rat adrenal cells

Forskolin, a unique diterpene which directly activates the adenylate cyclase, stimulated production of both cyclic AMP and corticosterone in isolated rat adrenal cells, in vitro. This agent also potentiated the action of adrenocorticotropin and/or cholera toxin on cyclic AMP production and steroidogenesis at lower concentrations. It augmented both an early (cyclic AMP production) and a late (steroidogenesis) action of the hormone in the adrenal gland.     

Control of nucleotide and erythroascorbic acid pools by cyclic AMP in Neurospora crassa

UDPglucuronic acid and erythroascorbic acid were identified in extracts of the fungus Neurospora crassa. The concentrations of these two compounds are estimated, in growing wild type N. crassa, to be about 0.10 and 0.28 μmol/ml of cell water, respectively. The pools of these two compounds are regulated by cyclic AMP in Neurospora, both being elevated in the cr-1, adenylate cyclase deficient mutant and both being lowered by exogenous cyclic AMP. The pools of these two compounds are also elevated on nitrogen deprivation. The pools of a large number of other nucleotides are not influenced by cyclic AMP. Possible relationships between the metabolism of UDPglucuronic acid and erythroascorbic acid are discussed. It was found that exogenous cyclic AMP was much more effective in influencing cultures grown at 30–37°C than those grown at 25°C. We suggest that higher temperatures may render Neurospora more permeable to a variety of different compounds.     

Effects of cyclic AMP and dibutyryl cyclic AMP on amino acid transport in the isolated rat ovary.

Prepubertal rat ovaries were incubated in medium containing the non-utilizable amino acids alpha-aminoisobutyric acid (AIB-14C) or 1-aminocyclo-pentane-carboxylic acid (cycloleucine-14C). The rate of uptake of the two amino acids was studied in the isolated ovaries after different incubation periods. Addition of 5mM cyclic AMP (cAMP) caused a slight stimulation of the AIB-transport but in higher concentrations (10-25 mM) an inhibition was noted. With dibutyrl cyclic AMP (dbcAMP) a dose-dependent increase was seen with 0.5-5 mM concentrations with no further effect of higher concentrations. Time course studies were performed with both AIB and cycloleucine in presence of 10 mM dbcAMP and increased uptake values were noted at each time studied (30-240 min). The phosphodiesterase inhibitor aminophyline in lower concentrations did not influence AIB-transport but 5-10 mM caused increased uptake values in the ovaries. The stimulatory action of dbcAMP on amino acid transport was augmented by a low concentration of aminophylline (0.5 mM). Experiments were in addition carried out in the presence of puromycin and under these circumstances it was still possible to enhance amino acid transport by addition of dbcAMP. The results are discussed in relation to earlier reported effects of gonadotropins on ovarian amino acid transport.     

Unsaturated fatty acid effect on cyclic AMP levels in human embryo lung fibroblasts.

The addition of arachidonic acid at 250 muM to cultures of human embryo lung fibroblasts (IMR-90) increases cellular cyclic AMP levels within 5 minutes to approximately 15-fold over basal. Other unsaturated fatty acids, 11, 14, 17-eicosatrienoic, linoleic, 8, 11, 14-eicosatrienoic and oleic also cause similar rapid elevation of cellular cyclic AMP. During this time interval, no detectable conversion of the added linoleic or arachidonic acids to prostaglandin is observed. These cells produce prostaglandins at measurable concentrations in response to treatment with ascorbic acid or bradykinin. Saturated fatty acids have no influence on cyclic AMP levels in these cells. This effect of unsaturated fatty acids on cellular cyclic AMP levels varies with the cell type. For example, smooth muscle and endothelial cells obtained from the calf pulmonary artery show very little or no increase in cellular cyclic AMP upon exposure to arachidonic acid.     

The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat liver cells

The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat hepatocytes was studied. In the presence of low levels (10–100 μM) of the cyclic nucleotide the initial rate of uptake was increased significantly, with a peak occurring at about 20 μM. In contrast, concentrations of dibutyryl cyclic AMP between 200 μM and 1 mM caused a significant decrease in the initial rate of uptake of the bile acid by the cells. Sodium-dependent transport of taurocholic acid was found to be enhanced by 20 μM dibutyryl cyclic AMP, but sodium-independent uptake appeared to be unaffected. Inhibition by 1 mM dibutyryl cyclic AMP, however, was found to occur in both the sodium-dependent and -independent components of the transport system. The initial rate of taurocholic acid uptake in hepatocytes incubated with 1.2 mM extracellular calcium was increased compared to that in calcium-depleted cells, and this increase was entirely due to enhanced sodium-dependent transport. 1.2 mM calcium and 20 μM dibutyryl cyclic AMP together did not stimulate the uptake rate to a greater extent either treatment alone. It is conclude that calcium and low levels of dibutyryl cyclic AMP alter the rate of taurocholic acid uptake by changing the flux of sodium in the hepatocytes. The inhibitory effect of 1 mM dibutyryl cyclic AMP was not relieved by the presence of 1.2 mM calcium in the cell incubation medium. The results show that dibutyryl cyclic AMP can affect the rate of transport of bile acid into liver cells, and suggest a possible regulatory role for cyclic AMP in this process.     

Export of cyclic AMP by mammalian reticulocytes

Suspensions of reticulocyte-enriched red cells produce and extrude cyclic AMP in proportion to their reticulocyte content. When reticulocytes are treated with the beta-adrenergic agonist isoproterenol, up to 3.5 pmoles of cyclic AMP/min/mg protein appear in the extracellular medium. Extruded cyclic AMP can occur against apparent concentration gradients and is inhibited by agents (iodoacetate, dinitrophenol) that deplete cellular ATP, as well as by probenecid and prostaglandin A1. Extrusion of cyclic AMP depends on the availability of intracellular cyclic AMP but is not obligatorily coupled to adenylate cyclase activity: extrusion continues following termination of a pulse of stimulation and exhibits a temperature dependence that differs from that for cyclic AMP production in response to isoproterenol. Erythropoietin affects neither production nor extrusion of cyclic AMP by reticulocytes. In whole blood, cyclic AMP extruded by reticulocytes may be a significant source of plasma cyclic nucleotide.     

Metabolism of cyclic AMP in non-pathogenic Mycobacterium smegmatis

The intracellular concentration of cyclic AMP reached a maximum in 3.5-day old cultures of Mycobacterium smegmatis grown in the presence of glycerol as the main source of carbon. Glucose-grown cells exhibited decreased cyclic AMP levels at all stages of growth. When M. smegmatis cells were incubated with various metabolites, pyruvate increased whereas glucose, citric acid, succinic acid and lactic acid decreased intracellular cyclic AMP levels. No cyclic AMP was detected in the incubation medium. The presence of a cyclic AMP-binding protein was demonstrated in cellfree extracts of M. smegmatis.     

Ultrastructural changes and cyclic AMP in frog oxyntic cells

In vitro frog gastric mucosa was employed as a model for a combined physiological, biochemical, and ultrastructural study of the morphological changes which accompany the onset of acid secretion by the oxyntic cell. The histamine H2-receptor antagonist metiamide was used to provide a reproducible control state. Stimulation of acid production by theophylline resulted in a 10-fold increase in plasma membrane surface area and a distinct change in the conformation of mitochondrial cristae. Studies using the acid secretion inhibitors, thiocyanate and anoxia, demonstrated that neither acid production per se nor oxidative metabolism is essential for the theophylline-dependent changes in surface area. Increases in tissue cyclic AMP levels were observed under the conditions producing morphological changes. It is postulated that surface area changes induced by theophylline are controlled by cellular cyclic AMP levels.     

Control of retinoic acid receptor expression in mouse melanoma cells by cyclic AMP

Retinoic acid receptor (RAR) α and γ mRNAs were constitutively expressed in B16 melanoma cells with or without retinoic acid (RA) treatment. RARβ mRNA, however, was significantly expressed only after exposure to RA. Induction of RARβ by RA occurred within 1 h and was not inhibited by cycloheximide (i.e., did not require new protein synthesis). All three RAR mRNA levels were dramatically decreased with 8-bromo-cyclic AMP treatment and could not be rescued by addition of RA. Analysis of RARγ revealed that this decrease occurred within 1 h of exposure to 8-bromo-cyclic AMP and was not blocked by simultaneous treatment with cycloheximide. The stability of RARγ mRNA was not altered by cyclic AMP treatment. Nuclear extracts from 8-bromo-cyclic AMP-treated cells showed a large decrease in protein binding to a retinoic acid response element (RARE) oligonucleotide compared to control cells. This correlated with a marked reduction of RA-stimulated RARE-reporter gene activity in transfected cells which were treated with cyclic AMP. Pretreatment of B16 cells with cyclic AMP prior to RA addition dramatically reduced induction of PKCα, an early marker of RA-induced cell differentiation. Thus, cyclic AMP can antagonize the action of RA most likely via its ability to inhibit RAR expression. © 1996 Wiley-Liss, Inc.     

On the role of cyclic AMP and cyclic GMP in steroid production by bovine cortical cells

The time course of corticotropin-induced steroidogenesis and changes in intracellular cyclic AMP and cyclic GMP levels were investigated in isolated bovine adrenocortical cells prepared by trypsin digestion. Corticotropin produced a pea a peak rise in cyclic AMP during the first 5 min of stimulation and enhanced steroid production after 15 min. Corticotropin also caused a decrease in cortical cyclic GMP at 5 min; this decrease in cyclic GMP reverted to a 2–3 fold increase at 15–30 min which gradually subsided by 60 min. A steroidogenic concentration of prostaglandin E₂ also produced an elevation in the levels of both nucleotides, but the rise in cyclic GMP preceded the rise incyclic AMP. These results suggest that the relative amount of cyclic AMP and cyclic GMP, rather than the absolute levels of cyclic AMP, may be a key factor in the regulation of steroidogenesis.     

A possible role of cyclic AMP on tracheary element formation in cultured carrot-root slices

When the root-phloem slices ofDaucus carota cv. Hokkaidô-gosun were cultured on a Murashige and Skoog's medium containing 2,4-dichlorophenoxyacetic acid (2,4-D medium) and cyclic AMP or its analogues, tracheary elements were formed in the dark, while they were not formed on the medium containing only 2,4-D in the dark. The number of tracheary elements induced by cyclic AMP was far less than that induced by cytokinin or 8-bromo-cyclic AMP. But when theophylline, an inhibitor of cyclic AMP phosphodiesterase, was used in combination with cyclic AMP in the culture, the number of tracheary elements was significantly increased. A remarkable increase in cytokinin activity was found in the hydrolyzate of soluble RNA extracted from the slices cultured on the 2,4-D medium containing 8-bromo-cyclic AMP, but only negligible cytokinin activity was detected in the hydrolyzate of soluble RNA extracted from the slices cultured on the 2,4-D medium without 8-bromo-cyclic AMP. Since cytokinin production occurred in the slices cultured in the light, it was supposed that light irradiation might induce cyclic AMP production. The mechanism of cytokinin production leading to tracheary element formation mediated by cyclic AMP level is discussed.     

On the role of cyclic AMP and cyclic GMP in steroid production by bovine cortical cells.

The time course of corticotropin-induced steroidogenesis and changes in intracellular cyclic AMP and cyclic GMP levels were investigated in isolated bovine adrenocortical cells prepared by trypsin digestion. Corticotropin produced a peak rise in cyclic AMP during the first 5 min of stimulation and enhanced steroid production after 15 min. Corticotropin also caused a decrease in cortical cyclic GMP at 5 min; this decrease in cyclic GMP reverted to a 2-3 fold increase at 15-30 min which gradually subsided by 60 min. A steroidogenic concentration of prostaglandin E2 also produced an elevation in the levels of both nucleotides, but the rise in cyclic GMP preceded the rise in cyclic AMP. These results suggest that the relative amounts of cyclic AMP and cyclic GMP, rather than the absolute levels of cyclic AMP, may be a key factor in the regulation of steroidogenesis.