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.     

Cyclic AMP-dependent protein kinase of Neurospora crassa

$\text{Neurospora}\text{crassa}$ was surveyed for cyclic AMP-dependent protein kinase activity. Two peaks (I and II) of protein kinase activity were demonstrated by DEAE-cellulose chromatography of wild type $\text{Neurospora}$ extracts. Peak I was stimulated by cyclic AMP, eluted below 60 mM NaCl and had high activity using histone H2B as substrate. Peak II eluted at 200–250 mM NaCl; its activity was not cyclic AMP stimulated and was highest with dephosphorylated casein as a substrate. Cyclic AMP binding to a protein associated with the protein kinase is specifically inhibited by certain cyclic AMP analogs.     

Cyclic AMP potentiates down regulation of epidermal growth factor receptors by platelet-derived growth factor

Pretreatment of $\text{Balb}\text{c}\text{-3T3}$ cells with platelet-derived growth factor (PDGF) has been shown to decrease binding sites for ¹²⁵I-labelled epidermal growth factor (EGF) (1,2,3). Agents which elevate cellular cyclic AMP concentrations enhance this ability, and the change in EGF binding is inversely proportional to the elevation of cyclic AMP. In quiescent density arrested cells, the sensitivity of cells to down regulation of EGF receptors by PDGF is proportional to the cyclic AMP content of the cultures in three different cell lines. Agents which elevate cyclic AMP and which potentiate PDGF mediated heterologous down regulation of EGF receptors are able, like cholera toxin (3), to stimulate cells to synthesize DNA in defined medium in the absence of EGF. Down regulation of EGF receptors by PDGF in combination with agents elevating cyclic AMP effectively mimics the action of EGF.     

Cyclic AMP levels in Drosophila during postembryonic development and in the adults.

The physiological content of Drosophila melanogaster tissues in cyclic AMP has been determined and its variations studied during postembryonic development and in the adults. Marked variations were observed, especially during metamorphosis where the ratio between the lowest and highest values (0·35 to 17·25 pmoles/mg protein) was $\text{1}\text{44}$. In larvae the variations of cyclic AMP level were not clearly related to the larval ecdyses, but the steps of metamorphosis, i.e. formation of the puparium, larval-pupal apolysis, and pupal-adult apolysis, were accompanied with rapid and drastic rises of cyclic AMP, up to the highest value mentioned. We therefore deduce that cyclic AMP is involved in the metamorphosis of D. melanogaster as a chemical signal. In adults, the cyclic AMP level was remarkably constant and was around 7 pmoles/mg protein.     

Interaction of cyclic nucleotides and ecdysterone in breaking the pupal diapause of the bertha armyworm, Mamestra configurata WLK.

Cyclic GMP and cyclic AMP have distinct and opposite effects upon the action of ecdysterone in diapausing pupae of the Bertha armyworm, $\text{Mamestra}$$\text{configurata}$. Cyclic GMP enhanced the effectiveness of suboptimal doses of ecdysterone in breaking diapause; the amount of cyclic GMP required to lower the ED50 of ecdysterone by half was 80 μg/g. Dibutyryl cyclic GMP had no apparent effect on the action of ecdysterone over a wide dose range (0.07 – 70 μg/g). On the other hand, cyclic AMP and dibutyryl cyclic AMP effectively blocked the diapause-breaking action of ecdysterone when administered simultaneously with the steroid hormone. The amount of cyclic AMP required to reduce the incidence of diapause termination from 100% to 50% was 60 μg/g; for dibutyryl cyclic AMP the amount required was only 14 μg/g. No cyclic nucleotide tested in the study could by itself break the pupal diapause of $\text{M.}$$\text{configurata}$. The concept that cyclic GMP and cyclic AMP provide at least different if not opposing regulatory influences in certain insect systems is discussed briefly in the light of these observations.     

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.     

Glucagon or cyclic AMP-stimulated synthesis of 5-phosphoribosyl 1-pyrophosphate in isolated hepatocytes and inhibition by antimicrotubular drugs.

Glucagon increased the level of 5-phosphoribosyl 1-pyrophosphate ($\text{PP}$Rib$\text{P}$) in isolated rat hepatocytes; a relatively high concentration of cyclic AMP could replace glucagon. In the presence of glucagon, the rate of incorporation of respective radioactive precursors into purine, pyrimidine, and oxidized pyridine nucleotides was accelerated, indicating that glucagon stimulates the synthesis of $\text{PP}$Rib$\text{P}$. Addition of 10^?6 M colchicine, vinblastin, or podophyllotoxin abolished the glucagon or cyclic AMP-induced increase in the $\text{PP}$Rib$\text{P}$ level. Colchicine did not affect accumulation of cyclic AMP induced by glucagon. These results suggest the involvement of tubulin or microtubules in the signal transfer from cyclic AMP to stimulated synthesis of $\text{PP}$Rib$\text{P}$.     

Modification of radiosensitivity of mammalian cells by cyclic nucleotides

Some $\text{in vitro}$ and $\text{in vivo}$ studies suggest that adesosine 3′,5′-cyclic monophosphate (cyclic AMP) may be one of the important factors in determining the radiosensitivity of certain mammalian cells; however, the role of guanosine 3',5'-cyclic monophosphate (cyclic GMP) in radiosensitivity of mammalian cells is completely unknown. Recent data also suggest that the mechanism of radiation protection afforded by moderate hypoxia and SH-containing compounds may involve an alteration in the intracellular level of cyclic AMP. At least one $\text{in vivo}$ study shows that cyclic AMP protects hair follicles and gut epithelial cells against radiation damage; however, it does not protect lymphosarcoma and breast carcinoma in mice. If a similar phenomenon is found in humans, an elevation of the intracellular level of cyclic AMP during radiation exposure may improve the effectiveness of radiation therapy in those cases where the radiation damage of normal tissue becomes the limiting factor for a continuation of the therapy program. More $\text{in vitro}$ and $\text{in vivo}$ studies on normal and cancer cells are needed to substantiate the role of cyclic nucleotides in radiosensitivity.     

Adenosine 3′, 5′-cyclic monophosphate in Schistosoma mansoni

Homogenates of adult $\text{Schistosoma mansoni}$ (blood flukes), isolated from the porto-mesenteric veins of infected mice, contain substantial activities of adenylyl cyclase, cyclic AMP phosphodiesterase, and a cyclic AMP stimulated protein kinase. The adenylyl cyclase, which is largely sedimentable at 10,000xg, is stimulated 20-fold by 10mM sodium fluoride and 1.4 to 2-fold by serotonin, glucagon, prostaglandins E₁, E₂ or B₁. The phosphodiesterase, which is largely sedimentable at 10,000xg, is inhibited by both aminophylline and papaverine but is not influenced by 10mM sodium fluoride. The protein kinase, which is present in the 10,000xg supernatant is stimulated 4 to 8-fold by either cyclic AMP or cyclic GMP. There is a preference for cyclic AMP ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.1×10}{}^{\mathrm{?7}}\text{M}$) over cyclic GMP ($\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 4.5×10}{}^{\mathrm{?6}}\text{M}$). If intact worms are incubated in a glucose free medium there is a mobilization of glycogen stores which is preceded by a rise in cyclic AMP concentration. In a medium with 5mM glucose there is neither a rise in cyclic AMP nor mobilization of glycogen.     

Dibutyryl cyclic AMP treatment mimics ovariectomy: new genomic regulation in mammary tumor regression

The $\text{in}\text{vitro}$ translated proteins from poly(A)RNAs differed when hormone-dependent mammary carcinomas were compared during their growth and regression. Within 6 hours post ovariectomy the concentration of one protein band increased and those of two protein bands decreased in the regressing as compared to the growing tumors. The translated protein patterns of the regressing tumors were identical whether regression was induced by ovariectomy or dibutyryl cyclic AMP treatment. The results suggest that mammary tumor growth is subject to genomic regulation and that the same new genetic event occurs in dibytyryl cyclic AMP- and ovariectomy-induced regression.     

Isolated adrenal cortex cells: ACTH agonists, partial agonists, antagonists; cyclic AMP and corticosterone production

Isolated adrenal cortex cells respond to the addition of ACTH_1–39 or analogs with increased production of cyclic AMP and corticosterone. It is estimated that cyclic AMP production need proceed at less than 20% of maximum to induce maximum corticosterone production. ACTH_1–24, [Lys¹⁷, Lys¹⁸]ACTH_1–8 amide, and ACTH_1–16 amide induce a maximum rate of cyclic AMP and of corticosterone production equal to those of ACTH_1–39. The relative potencies as determined by cyclic AMP and by corticosterone production are in excellent agreement. The analog, ACTH_5–24, induces maximum cyclic AMP production equal to 45% of that of the natural hormone, but as predicted, induces maximum corticosterone production equal to that of ACTH_1–39. The derivative, [Trp(Nps)⁹]ACTH_1–39 induces 77% of maximum corticosterone production and less than 1% of maximum cyclic AMP production. The fragment ACTH_11–24 is a competitive antagonist of ACTH_1–39 for both cyclic AMP and corticosterone production. The observations on agonists, a partial agonist and a competitive antagonist are in harmony with the “second messenger” role assigned to cyclic AMP. A provisional model, based on the fit of the experimental observations to a set of equations, provides expressions of “intrinsic activity,” “receptor reserve”, “sensitivity”, and “amplification” in terms of maximum cyclic AMP production, concentration of ACTH which induces $\text{1}\text{2}$ maximum cyclic AMP production and concentration of cyclic AMP which induces $\text{1}\text{2}$ maximum corticosterone production.     

Uterine cyclic AMP formation: biphasic effect of estrogen on catecholamine sensitivity.

Female, ovariectomized rats were treated with estradiol and then, after various time periods, given an intravenous injection of isoproterenol or epinephrine. 30 seconds later uteri were frozen $\text{in}$$\text{situ}$ and assayed for cyclic AMP and glycogen phosphorylase. The cyclic AMP response to catecholamines was significantly depressed as early as 30 minutes after estrogen and at 6, 12 and 24 hours was 50% of that in non-estrogen-treated controls. Catecholamine-induced glycogen phosphorylase activation was unchanged until 24 hours after estrogen when it was significantly increased over controls. At 48 hours of estrogen both the cyclic AMP and phosphorylase responses to catecholamines were greater than controls. Estrogen regulates uterine β-adrenergic sensitivity but the time courses of estrogen effects on the cyclic AMP and glycogen phosphorylase response changes are different. Catecholamine-induced uterine cyclic AMP formation is biphasic: suppression during the first 24 hours of estrogen followed by recovery and finally augmentation by 48 hours. Catecholamine-induced glycogen phosphorylase activation shows only augmentation after 24–48 hours of estrogen. It is concluded that estrogen has independent effects on the β-adrenergic-glycogen phosphorylase activation pathway at two different points; one prior to cyclic AMP formation and another after cyclic AMP formation.     

Effects of choleragen on hormonal responsiveness of adenylate cyclase in human fibroblasts and rat fat cells

Choleragen increases cyclic AMP content of confluent human fibroblasts. Maximally effective concentrations of isoproterenol and prostaglandin E₁ also induce large increases in cyclic AMP content of human fibroblasts and in confluent cultures the effect of prostaglandin E₁ is much greater than that of isoproterenol. After incubation with choleragen, the increment in cyclic AMP produced by 2 μM isoproterenol is increased and approaches that produced by 5.6 μM prostaglandin E₁. Although the concentration of isoproterenol which produces a maximal increase in cyclic AMP is similar in both control and choleragen-treated cells, lower concentrations of isoproterenol are more effective in the choleragen-treated cells. In choleragen-treated cells, although the response to 5.6 μM prostaglandin E₁ is reduced by as much as 50%, the concentration of prostaglandin E₁ required to induce a maximal increase in cyclic AMP is $\text{1}\text{10}$ that required in control cells. Thus the capacities of intact human fibroblasts to respond to isoproterenol and prostaglandin E₁ can be altered independently during incubation of intact cells with choleragen. Differences in responsiveness to the two agonists were not demonstrable in adenylate cyclase preparations from control or choleragen-treated cells.In rat fat cells, the effects of choleragen on cyclic AMP content were much smaller than those in fibroblasts. In contrast to its effect on intact fibroblasts, choleragen treatment of rat fat cells did not alter the accumulation of cyclic AMP in response to a maximally effective concentration of isoproterenol. The responsiveness of adenylate cyclase preparations to isoproterenol was also not altered by exposure of fat cells to choleragen.     

Genetic manipulation of cyclic AMP levels in Drosophila melanogaster.

Cyclic AMP levels in $\text{Drosophila}$,$\text{melanogaster}$ adults can be altered genetically by changing the number of doses of chromomere 3D4 contained in the genome, a chromomere previously shown to control the activity of cyclic AMP phosphodiesterase in a dose-dependent manner. Flies completely deficient for chromomere 3D4 have 2–7 times the cyclic AMP level of flies with one or two doses of chromomere 3D4. Cyclic AMP levels are significantly depressed in flies carrying three doses of 3D4. Cyclic GMP levels are not influenced in a dose-dependent manner by chromomere 3D4. The effect on cyclic AMP levels may provide a useful system for investigating physiological and developmental consequences of aberrant cyclic AMP levels in the intact organism.     

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.     

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.     

The influence of adenosine 3′,5′-monophosphate upon the activity of the membrane-associated (Ca2++Mg2+)-dependent adenosine triphosphatase of the human erythrocyte

The phosphohydrolase activity of the membrane-associated $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+Mg}{}^{\mathrm{2+}}\text{)-dependent}$ adenosine triphosphatase (ATPase) of the human erythrocyte can be inhibited by micromolar or nanomolar concentrations of cyclic AMP. Millimolar concentrations of cyclic AMP are less effective. The inhibitory effect of cyclic AMP is potentiated in the presence of the phosphodiesterase inhibitor, theophylline.     

Adenosine-elicited accumulation of cyclic AMP in brain slices: potentiation by agents which inhibit uptake of adenosine

The uptake and incorporation of low concentrations of radioactive adenosine into guinea pig cerebral cortical slices is effectively inhibited by dipyridamole, hexobendine, papaverine, 6-($\text{p}$-nitrobenzylthio) guanosine, 5′-deoxy-adenosine and N⁶-phenyladenosine and ineffectively inhibited by other adenosine analogs such as 2-chloroadenosine, 3′-deoxyadenosine and tubercidin or by phosphodiesterase inhibitors such as theophylline, isobutylmethylxanthine, and N, 0-dibutyrylcyclic AMP. When uptake of 10–20

adenosine is inhibited 50–70% by dipyridamole, hexobendine, papaverine or 6-($\text{p}$-nitrobenzylthio)-guanosine, the adenosine-elicited accumulation of cyclic AMP is potentiated 2–3 fold. Potentiation of the effects of low concentrations of adenosine by various agents parallels more closely their efficacy as inhibitors of adenosine uptake rather than their potency as phosphodiesterase inhibitors. Amine-elicited accumulations of cyclic AMP are enhanced by hexobendine, dipyridamole, papaverine and 6-($\text{p}$-nitrobenzylthio) guanosine and this enhancement is blocked by an adenosine antagonist, theophylline. The stimulatory effects of the adenosine analogs, 5′-deoxyadenosine, 2-chloroadenosine and N⁶-phenyladenosine are blocked by theophylline and potentiated by hexobendine. The results are compatible with the hypothesis that the specific inhibition of uptake of adenosine potentiates adenosine or amine-elicited accumulations of cyclic AMP by increasing the effective extracellular concentration of adenosine within the slice. The inhibition or stimulation of cyclic AMP accumulation by adenosine analogs is consonant with differential activities as agonist or antagonist at an extracellular adenosine receptor.     

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 $\text{in vivo}$.     

The role of cyclic AMP in the thermosensitive lesion of the formation of closed covalent circular Rts 1 DNA.

The formation of closed covalent circular DNA of R factor, Rts 1, does not take place at non-permissive temperature, 42°C, in $\text{E. coli}$ 20SO. However, when Rts 1 was placed in mutants having a low level of cyclic AMP or lacking cyclic AMP receptor protein, the thermosensitive lesion is overcome. Addition of cyclic AMP caused inhibition of the formation of ccc DNA in mutants with low cyclic AMP level, but not in mutants lacking cyclic AMP receptor protein.