Abnormally high rate of cyclic AMP excretion from an Escherichia coli mutant deficient in cyclic AMP receptor protein

By labeling adenosine 3′, 5′-cyclic monophosphate (cyclic AMP) with [³²P] phosphate and chromatographing it on a thin-layer alumina plate, we have determined the extra- and intracellular amounts of cyclic AMP in an $\text{Escherichia coli}$ CRP^? mutant (deficient in a cyclic AMP receptor protein) and its isogenic CRP⁺ cell. The CRP^? cell was found to excrete cyclic AMP at an abnormally high rate as compared to the CRP⁺ cell when growing on glucose or glycerol, which can be correlated with the abnormally high intracellular levels of cyclic AMP in the CRP^? cell.     

In vivo activation of cyclic adenosine 3':5'-phosphate-dependent protein kinase in Chinese hamster ovary cells treated with N-6, O-2'-diburyl cyclic adenosine 3':5'-phosphate.

Treatment of Chinese hamster ovary cells with dibutyryl cyclic AMP, which results in a net increase of the intracellular cyclic AMP level, converts the epithelial-like cells to a fibroblast-like shape. Protein kinase activity in cells treated with 1 mM dibutyryl cyclic AMP show a 3-fold increase in V_max but no appreciable changes in the apparent K_m for ATP. When cells are treated with dibutyryl cyclic AMP, there is a time-dependent conversion of cyclic AMP-stimulable protein kinase to cyclic AMP-independent catalytic subunits, as demonstrated by Sephadex G-100 gel filtration. These experiments demonstrate the activation of the cyclic AMP-dependent protein kinase $\text{in vivo}$. This activation may lead to phosphorylation of certain cellular constituent(s) and thus may be involved in the observed morphological transformation.     

Effect of O2-monobutyryl guanosine 3', 5'-cyclic monophosphate on hepatic metabolism of free fatty acids.

Livers from fed male rats were perfused $\text{in vitro}$ with O^2′-monobutyryl guanosine 3′,5′-cyclic monophosphate. The output of triglyceride was reduced, while output of ketone bodies and glucose was stimulated by 10^?4M monobutyryl guanosine 3′,5′-cyclic monophosphate. No effect was observed with 10^?5 M nucleotide. Monobutyryl guanosine 3′,5′-cyclic monophosphate did not affect uptake of free fatty acids. In these respects, monobutyryl guanosine 3′,5′-cyclic monophosphate mimics the effects of dibutyryl adenosine 3′,5′-cyclic monophosphate, although the guanylic nucleotide seems to be less potent than the adenosine 3′,5′-cyclic monophosphate derivative.     

In vitro stimulation of tadpole tail regression by cyclic AMP.

Treatment of $\text{Rana catesbeiana}$ tail fin tissue $\text{in vitro}$ with 0.1 mM or 0.5 mM cyclic AMP or with triiodothyronine induces an increase in the specific activity of hexosaminidase, a lysosomal marker enzyme, and a decrease in tissue area. Lithium chloride (8 mM), an inhibitor of adenylate cyclase, inhibits these changes when initiated by triiodothyronine but not when initiated by cyclic AMP. The levels of cyclic AMP, determined by radioimmunoassay techniques, increased 110 ± 10% over matched discs in culture after only one day's exposure to triiodothyronine. These results indicate the effect of triiodothyronine on fin resorption may be mediated by cyclic AMP.     

Effect of morphine sulfate on adenylate cyclase and phosphodiesterase activities in rat corpus striatum.

The effect of morphine sulfate (MS) on adenylate cyclase (AC) and phosphodiesterase (PDE) activities in the rat striatum was investigated. MS produced a dose-dependent increase in basal AC activity and did not alter sodium fluoride-induced stimulation both $\text{in}$$\text{vivo}$ (7.5–30 mg/kg, 1 hr pretreatment, i.p.) and $\text{in}$$\text{vitro}$ (1–100μM). $\text{in}$$\text{vitro}$, when submaximal effective concentrations of dopamine and MS were combined, there was an additive effect. However, administration of MS $\text{in}$$\text{vivo}$ did not alter dopamine-induced stimulation of AC activity. MS, $\text{in}$$\text{vitro}$ and $\text{in}$$\text{vivo}$ inhibited PDE activity in a dose-dependent manner only with the high substrate concentration (3.3 × 10⁻³M cyclic AMP). Preliminary results from this study indicate that morphine affects the cyclic AMP system.     

Cyclic AMP-induced tyrosinase synthesis in Neurospora crassa

Cyclic AMP induces the synthesis of tyrosinase in $\text{Neurospora crassa}$. Adenine, adenosine, 3′-AMP, 5′-AMP, and 2′,3′-cyclic AMP have no inductive effect while 8-bromocyclic AMP and dibutyryl cyclic AMP are good inducers. Caffeine and theophylline, inhibitors of cyclic AMP phosphodiesterase, also induce tyrosinase. A possible relationship between cyclic AMP induction and previously reported induction by cycloheximide is suggested.     

A cyclic AMP dependent protein kinase in Dictyostelium discoideum

A cyclic AMP-dependent protein kinase was found to appear during the time course of development of $\text{Dictyostelium}\text{discoideum}$. No cyclic AMP dependency was observed at any stage of development in crude 110,000 X G soluble extracts. After partial purification, however, extracts from post-aggregation stages contained enzyme that was activated up to 6-fold by cyclic AMP, whereas protein kinase from earlier stages was not affected by cyclic AMP. Likewise, cyclic AMP binding activity increased from the aggregation to the slug stage of development. Approximately one-half of the total cyclic AMP binding activity co-purified with the cyclic AMP dependent protein kinase. The enzyme from $\text{Dictyostelium}$ showed similarities to mammalian protein kinases with respect to its kinetic properties but differed in its behavior on ion-exchange chromatography.     

Regulation of the basal and cyclic AMP-stimulated rates of glycogen synthesis in Escherichia coli by an intermediate of purine biosynthesis

In $\text{Escherichia}\text{coli}$an abrupt increase in the rate of glycogen synthesis occurs at the onset of total nitrogen starvation. We present here both $\text{in}\text{vivo}$ and $\text{in}\text{vitro}$ data indicating that this increase occurs because of the loss of a nitrogen-containing intermediate of purine biosynthesis (apparently 5-aminoimidazole-4-carboxamide ribonucleotide) that inhibits glycogen synthesis. We also show that this inhibitory intermediate antagonizes the stimulation of glycogen synthesis by 3′,5′-cyclic AMP. The uncovering of the regulation of glycogen synthesis by this inhibitor apparently provides the first link in understanding the 23-year-old observation of a reciprocal relationship between growth rate and glycogen accumulation in $\text{E.}\text{coli}$.     

The effect of cyclic AMP on glycoprotein secretion in isolated rat hepatocytes

The effects of dibutyryl cyclic AMP on glycoprotein biosynthesis, intracellular mobilization, and secretion in isolated rat hepatocytes are described. Dibutyryl cyclic AMP (2.5 mm) initially suppresses [³H]glucosamine or [³H]fucose incorporation into cellular macromolecular material; however, after $\text{3}\text{1}\text{2}$ h, the incorporation of these radiolabeled carbohydrates into macromolecular material was stimulated relative to control cells. The stimulation in accumulation of cellular glycoprotein occurred in membrane-associated fractions, with most of this accumulation occurring in the Golgi elements. The glycoprotein produced in the presence of dibutyryl cyclic AMP was quantitatively precipitated by antibodies directed against rat serum, suggesting that the accumulated cellular material is normally destined for secretion from the cell. Dibutyryl cyclic AMP also produced a drastic inhibition of glycoprotein secretion which persisted during the cellular accumulation of glycosylated material. Exposure of the hepatocytes to colchicine (10 μm) produced a similar increase in accumulation of [³H]glucosamine-containing immunoprecipitable material in the cellular fraction and a similar inhibition in secretion. The initial dibutyryl cyclic AMP-mediated suppression of synthesis of intracellular glycosylated material occurred entirely in non-membrane-associated intracellular fractions. Also, the initial accumulation of [³H]glucosamine-containing immunoprecipitable material was not suppressed during the first $\text{3}\text{1}\text{2}$ h after exposure to dibutyryl cyclic AMP, suggesting the initial suppression represents a metabolic process unrelated to secretion. The incorporation of [³H]leucine into macromolecular material was inhibited in both cellular and secreted fractions after exposure to dibutyryl cyclic AMP; however, the accumulation into the extracellular environment was inhibited to a greater extent. The patterns of [³H]glucosamine-containing lipid biosynthesis were unaffected by dibutyryl cyclic AMP.     

Cyclic AMP production in isolated rat seminiferous tubule cell preparations: A potential in vitro assay for follicle stimulating hormone

A simple procedure for the isolation of rat seminiferous tubule cell preparations free of Leydig cells is described. Both ovine and human FSH stimulated cyclic AMP accumulation. in proportion to the concentration of the hormones. Other hormones, including ICSH and hCG, were inactive. It is proposed that stimulation of cyclic AMP accumulation in tubule cells may be a specific, sensitive and rapid $\text{in vitro}$ assay for FSH.     

Inability of detect cyclic AMP in vegetative or sporulating cells or dormant spores of Bacillus megaterium

Cyclic AMP was not found in vegetative cells or sporulating cells or dormant spores of $\text{Bacillus}\text{megaterium}$ using an assay which would have detected an $\text{in}\text{vivo}$ concentration of 1 – 2 × 10^?9 M. Adenyl cyclase and cyclic AMP phosphodiesterase were also not detected in sonicates of vegetative or sporulating $\text{B.}\text{megaterium}$ cells.     

Adenosine 3', 5'-monophosphate in mycobacteria.

Cyclic adenosine 3′,5′-monophosphate (cyclic AMP) is present in saprophytic fast growing as well as pathogenic and non-pathogenic slow growing mycobacteria. Apparently there does not seem to be any direct relationship between either intra- or extra-cellular cyclic AMP content with the growth rate of the bacteria. Intracellular cyclic AMP content is much higher than that of $\text{E}$. $\text{coli}$ grown on a similar carbon source. Glucose when added to the cells suspended in phosphate buffer lowers the intracellular cyclic AMP content by 6–8 fold.     

Effects of hormones and nucleotides on ciliary beating in frog esophagus and guinea pig trachea

The $\text{in vivo}$ effects of various hormones, nucleotides and related substances on the rate of ciliary beating in frog esophagus and guinea pig trachea were studied using both particle transport and photoelectric methods. Frog esophageal ciliary beating $\text{in vitro}$ was greatly accelerated by acetycholine, eserine, prostaglandin A₁ and E₂, N⁶- 2′-0-dibutyryl-adenosine--3′,5′-cyclic monophosphate, all tri- and di-phosphonucleotides (ATP, ADP, UTP, UDP, etc.), EDTA, EGTA, and calcium-free medium. Adenosine monophosphate, epinephrine, serotonin at low concentrations, 3,3′5-L-triiodothyronine, creative phosphate, and phosphoenolpyruvate were inactive or only minimally stimulatory in the frog. All substances tested, including those trachea. Furthermore, guinea pig ciliary activity was unaffected by hyperthyroidism or hypothyroidism induced in the intact animal before testing.     

Effectors of lipoprotein lipase secretion from isolated cardiac muscle cells incubated in vitro

Cycloheximide, colchicine, tunicamycin, glucagon, dibutyryl-3′–5′-cyclic AMP, dexamethasone and hydrocortisone had no effect on the lipoprotein lipase activity associated with rat cardiac muscle cells incubated $\text{in vitro}$. However, the steroid hormones and inhibitors affected profoundly the appearance of extracellular enzyme during the incubations. The pattern of effects, was consistent with lipoprotein lipase being a normal secretory product of heart muscle cells.     

The estrogenic activity of DDT: Invivo and invitro induction of a specific estrogen inducible uterine protein by o,p'-DDT

The pesticide o,p'-DDT stimulates the production of a specific uterine protein, the so-called induced protein or IP, normally associated with an estrogenic response of the uterus. $\text{In}$$\text{vivo}$ stimulation of IP production is observed 1 hour after the administration of 250 mg/kg of o,p'-DDT to immature rats. $\text{In}$$\text{vitro}$ stimulation of IP production is observed after a 1 hour incubation of uteri with 100 μM o,p'-DDT. This $\text{in}$$\text{vitro}$ response is blocked by Actinomycin D. In contrast to o,p'-DDT, which binds to the cytoplasmic estrogen receptor and stimulates IP production, p,p'-DDT which does not bind well to the estrogen receptor does not stimulate IP production $\text{in}$$\text{vitro}$. These findings represent the first report of an estrogenic effect of o,p'-DDT in a completely $\text{in}$$\text{vitro}$ system.     

Cyclic AMP-dependent inactivation of human liver pyruvate kinase.

Human liver pyruvate kinase is rapidly (within 2 min) inactivated by incubation of a human liver supernatant with cyclic AMP, when measured at suboptimal substrate concentrations. Half-maximal inactivation is reached with 0.04 μM cyclic AMP. The apparent K_0.5 for phosphoenolpyruvate shifts from 0.5 mM to 1.1 mM by incubation with cyclic AMP. It is concluded that cyclic AMP-dependent protein kinase may catalyze the phosphorylation of human liver pyruvate kinase $\text{in vivo}$.     

Morphologic Differentiation of Mouse Neuroblastoma Cells induced <Emphasis Type="Italic">in vitro</Emphasis> by Dibutyryl Adenosine 3′:5′-Cyclic Monophosphate

The mechanism of mammalian neural differentiation is still obscure; but the availability of mouse neuroblastoma cells in vitro provides an opportunity to study some possible inducers of differentiation and this may help to elucidate the events involved at the molecular level. We have reported¹ that X-irradiation of mouse neuroblastoma cells in vitro induces the formation of axons. The differentiated cells seem to undergo maturation: the soma and nucleus increase in size and the cytoplasm becomes granular. Here we report that N⁶O² dibutyryl adenosine 3′:5′-cyclic monophosphate (dibutyryl cyclic AMP) induces axon formation in mouse neuroblastoma cells in vitro.     

The effect of cyclic AMP on anaerobic growth of Escherichia coli

Adenosine 3′,5′-cyclic phosphate (cyclic AMP) stimulated a cyclic AMP-deficient mutant strain of $\text{Escherichia coli}$ to grow anaerobically on glucose in a minimal medium and in media supplemented with nitrate or casein hydrolysate. Cyclic AMP was found to stimulate the production of the formic hydrogenlyase system in this mutant strain, but had no effect on its ability to carry out anaerobic reductions of nitrate or nitrite. It was also observed that CO₂ stimulated the anaerobic growth of the mutant in the absence of cyclic AMP.