In vitro Tn7 mutagenesis of Haemophilus influenzae Rd and characterization of the role of atpA in transformation.

Haemophilus influenzae Rd is a gram-negative bacterium capable of natural DNA transformation. The competent state occurs naturally in late exponential growth or can be induced by a nutritional downshift or by transient anaerobiosis. The genes cya, crp, topA, and sxy (tfoX) are known to function in the regulation of competence development. The phosphoenolpyruvate:carbohydrate phosphotransferase system functions to maintain levels of cyclic AMP necessary for competence development but is not directly involved in regulation. The exact signal(s) for competence and the genes that mediate the signal(s) are still unknown. In an effort to find additional regulatory genes, H. influenzae Rd was mutated by using an in vitro Tn7 system and screened for mutants with a reduced ability to induce the competence-regulatory gene, comA. Insertions in atpA, a gene coding for the alpha subunit of the F1 cytoplasmic domain of the ATP synthase, reduce transformation frequencies about 20-fold and cause a significant reduction in expression of competence-regulatory genes, while the expression of constitutive competence genes is only minimally affected. In addition, we found that an insertion in atpB, which encodes the a subunit of the F0 membrane-spanning domain, has a similar effect on transformation frequencies.     

Natural transformation and DNA uptake signal sequences in Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans is a member of the family Pasteurellaceae and a major causative agent of periodontitis. While several genera from this family are known to be competent for transformation, A. actinomycetemcomitans has yet to be fully characterized. Here we show that the competence of A. actinomycetemcomitans is remarkably similar to that of Haemophilus influenzae. In addition to having a similar frequency of transformation as H. influenzae, A. actinomycetemcomitans competence could also be induced at least 100-fold by cyclic AMP, suggesting that, as in H. influenzae, at least some competence genes are regulated by catabolite repression. Even more intriguing was the discovery of a putative A. actinomycetemcomitans DNA uptake signal sequence (USS) virtually identical to the USS of H. influenzae. Moreover, we provide evidence that this sequence functions in the same capacity as that from H. influenzae; the sequence appears to be required and sufficient for DNA uptake in a variety of assays. Finally, we have taken advantage of this system to develop a simple, highly efficient competence-based method for generating site-directed mutations in the wild-type fimbriated A. actinomycetemcomitans.     

Cyclic AMP specifically blocks proliferation of rat 3T3 cells transformed by polyomavirus.

Elevated exogenous and intracellular levels of cyclic AMP could totally block proliferation of polyomavirus (PyV) transformants derived from rat 3T3 cells without affecting proliferation of normal cells or simian virus 40 (SV40)-induced transformants. Concanavalin A (ConA) had the opposite effect; it could totally block proliferation of both normal cells and SV40 transformants but reduced proliferation of PyV transformants only twofold. Adenylate cyclase was threefold less active in membranes of PyV transformants, and the number of ConA receptors was similar to that of normal cells. Proliferating PyV transformants contained threefold less cyclic AMP than did proliferating SV40 transformants. The sensitivity to cyclic AMP did not correlate with the degree of transformation: cells transformed by Rous sarcoma virus and tumor cells derived from SV40 transformants were not sensitive to cyclic AMP. The differential effect of cyclic AMP and ConA on proliferation was probably due to the activity of an intact middle t protein. The presence of both large T and small t together with middle t was also required for cyclic AMP sensitivity.     

Neurotensin regulation of TSH secretion in the rat

The ionophore A23187 (6.7 microM) increased the rates of formation of prostaglandins and cyclic AMP in suspensions of thioglycollate-elicited rat peritoneal macrophages. Both effects were inhibited by the calmodulin blocker trifluoperazine (50 microM) and the calcium channel blocker verapamil (500 microM). Inhibitors of phospholipase A2 and cyclo-oxygenase also blocked both actions of A23187. The stimulated prostaglandin formation was markedly reduced when the cells were preincubated with 8-bromo-cyclic AMP (1mM), dibutyryl cyclic AMP (1mM) or cholera toxin (500ng/ml). Addition of exogenous arachidonic acid (30 microM) alleviated this inhibition. We propose that the effect of A23187 on macrophages includes a 'self-limiting' mechanism whereby newly-synthesized prostaglandins can inhibit, via cyclic AMP, a step(s) prior to the transformation of arachidonic acid and thus modulate their own production.     

Metabolic regulation of steroidogenesis in isolated adrenal cells of rat. Relationship of adrenocorticotropin-, adenosine 3':5'-monophosphate-and guanosine 3':5'-monophosphate-stimulated steroidogenesis with the activation of protein kinase.

The data presented with the isolated adrenal cells, in the present study, show that adrenocorticotropin in the physiological concentration range stimulates the synthesis of guanosine 3':5'-monophosphate(cyclic GMP), protein kinase activity, and steroidogenesis in a concentration-dependent manner without detectable rise in the levels of adenosine 3':5'-monophosphate (cyclic AMP). Millimolar concentrations of cyclic AMP and cyclic GMP, which stimulate corticosterone synthesis, also activate kinase activity and steroidogenesis in a sigmoid concentration-response manner. The process of phosphorylation activated by corticotropin, cyclic AMP and cyclic GMP is not inhibited by cycloheximide or actinomyin D. It is therefore proposed that the hormonal responses mediated by cyclic GMP and cyclic AMP are via the protein kinase enzymatic steps, and the inhibitory effect of cycloheximide and actinomycin D in corticotropin-stimulated steroidogenesis follows this step. In conjuction with our previous observations that the biosynthetic steps from (20S)-20-hydroxycholesterol to corticosterone are neither inhibited by cycloheximide nor affected by cyclic GMP, it is inferred that the rate-limiting step of adrenal steroidogenesis is the transformation of cholesterol to (20S)-20hydroxycholesterol and this very step is regulated by cyclic GMP and cyclic AMP. Of further significance are the findings that micromolar cincentrations of cyclic AMP and cyclic GMP, which do not stimulate steroidogenesis, effectively stimulate protein kinase activity in a concentration-dependent manner. It is therefore concluded that all cyclic-nucleotide-dependent protein kinase activities of the cell are not necessarily related to steroidogenesis.     

Prostaglandin stimulation of adenosine 3',5'-monophosphate accumulation in cultured chondrocytes in the presence or absence of parathyroid hormone

The effect of prostaglandin analogues on the cyclic AMP level in cultured chondrocytes were examined. Prostaglandin E1 at 0.4 to 30 microM, increased the intracellular concentration of cyclic AMP in chondrocytes. Its effect was rapid, being evident within 1 min and reaching a maximum in 10 to 20 min. The maximum level was sustained until 30 min after its addition and then decreased gradually. Prostaglandin D2 and E2 also increased the cyclic AMP level in chondrocytes, but they had less effect than prostaglandin E1. Prostaglandin A1 had no effect on the nucleotide level in chondrocytes, although they markedly increased the level in fibroblasts. The time course of stimulation of cyclic AMP accumulation in chondrocytes by prostaglandin E1, D2 or E2 was quite different from that by parathyroid hormone (PTH): the effect of prostaglandin was slower and more sustained than that of PTH. PTH potentiated the effect of prostaglandin E1, E2, or D2 on the cyclic AMP level in chondrocytes and that the combined effects of prostaglandin and PTH were more than additive. Addition of an inhibitor of cyclic nucleotide phosphodiesterase with prostaglandin, PTH or both produced a synergistic effect on the accumulation of cyclic AMP in the chondrocytes. These findings suggest that prostaglandin E1, E2, and D2 increase the synthesis of cyclic AMP and that the combined effect of the prostaglandins and PTH on the cyclic AMP level in chondrocytes is partly attributed to the synergistic synthesis of cyclic AMP in the cells.     

The role of cyclic AMP in the chemotactic responsiveness and spontaneous motility of rabbit peritoneal neutrophils. The inhibition of neutrophil movement and the elevation of cyclic AMP levels by catecholamines, prostaglandins, theophylline and cholera toxin.

Agents known to affect intracellular levels of cyclic AMP in many diverse systems have been tested for their effect on the chemotaxis induced by Escherichia coli culture filtrates, spontaneous motility and cyclic AMP levels of rabbit peritoneal neutrophils. Prostaglandin E1 and A1 but not prostaglandin F2alpha increased neutrophil cyclic AMP levels and, correspondingly, only the former two prostaglandins inhibited chemotaxis. Nevertheless, a quantitative relationship between prostaglandin stimulation of cyclic AMP and inhibition of chemotaxis could not be found. Epinephrine, isoproterenol, and, to a much lesser extent, norepinephrine increased neutrophil cyclic AMP through beta adrenergic stimulation. Only epinephrine and isoproterenol inhibited chemotaxis, but the inhibition was variable and not related to the ability of these catecholamines to increase intracellular cyclic AMP. Cholera toxin increased neutrophil cyclic AMP after a 30-min lag period which paralled its inhibitory effect on chemotaxis and spontaneous motility. However, the effect on chemotaxis require 50 ng/ml of toxin whereas the effect on cyclic AMP was manifested at 2 ng/ml of toxin. Prior to 30-min preincubation there was no effect of even 1250 ng/ml of toxin on either cyclic AMP or chemotaxis. Choleragenoid prevented the effects of toxin on both cyclic AMP and chemotaxis. The bacterial chemotactic factor obtained from E. coli culture filtrates did not effect a measurable change in levels of neutrophil cyclic AMP. The data indicate that even though cyclic AMP is not, in the main sequence of events, triggering the chemotactic response, increases in neutrophil cyclic AMP may modulate the movement and thus the chemotactic responsiveness of the neutrophil.     

Receptor-mediated gonadotropin action in the ovary. Inhibitory actions of concanavalin A and wheat-germ agglutinin on gonadotropin-stimulated cyclic AMP and progesterone responses in ovarian cells.

Pretreatment of ovarian cells with concanavalin A and wheat-germ agglutinin blocked the gonadotropin-induced cyclic AMP and progesterone responses and this effect was time- and concentration-dependent. Basal production of either cyclic AMP or progesterone, however, was not affected by treatment of cells with lectin. The effect of concanavalin A on gonadotropin-mediated cyclic AMP and progesterone responses was blocked by alpha-methyl D-mannoside and alpha-methyl d-glucoside. Similarly the inhibitory effect of wheat-germ agglutinin was reversed by N-acetyl-D-glucosamine. Pretreatment of ovarian cells with concanavalin A or wheat-germ agglutinin had no effect on protein synthesis in the ovary as monitored by [3H]proline incorporation studies. Concanavalin A and wheat-germ agglutinin did not affect steroid production in response to dibutyryl cyclic AMP and 8-bromo cyclic AMP, indicating that the inhibitory action of lectin was occurring at a step before cyclic AMP formation. Lectins specific for L-fucose, D-galactose and N-acetyl-D-galactosamine, gorse seed agglutinin, peanut agglutinin and Dolichos biflorus agglutinin respectively, did not interfere with gonadotropin-induced cyclic AMP and progesterone responses. The present studies suggest that gonadotropin receptors may be glycoprotein in nature or closely associated with glycoprotein structures with the carbohydrate chain containing N-acetyl-D-glucosamine, mannose and possibly N-acetylneuraminic acid.     

Activity of imidazole on the hydrolysis of cyclic AMP and cyclic GMP by bovine heart and rat liver cyclic nucleotide phosphodiesterases.

The effects of imidazole on the hydrolysis of cyclic AMP and cyclic GMP by crude and partially purified phosphodiesterases obtained from bovine heart and rat liver were studied in order to determine if imidazole has an activity on cyclic nucleotide hydrolysis under conditions which might explain its ability to antagonize the effects of several hormones. Imidazole-Cl (40 mm, pH 7.4) had no effect on the hydrolysis of cyclic AMP or cyclic GMP at substrate levels below 10 μm by the crude enzymes but increasing stimulation was observed with increasing substrate concentrations reaching a twofold stimulation at 1 mm cyclic nucleotide. Three phosphodiesterases with varying substrate specificities were partially purified from bovine heart by ammonium sulfate precipitation and diethyl aminoethyl cellulose chromatography. With these enzymes imidazole had less stimulatory activity and some inhibitory effect on the hydrolysis of 10^?4m cyclic AMP and cyclic GMP but was without significant effect on the hydrolysis of 10^?6m cyclic AMP or cyclic GMP. The stimulatory activity of imidazole on the hydrolysis of high levels of cyclic nucleotide was dependent on the presence of phosphodiesterase activator. The stimulatory effect of the activator and imidazole plus activator on the hydrolysis of 10^?4m cyclic GMP by the rather cyclic GMP-specific enzyme could be eliminated by the addition of ethylene glycol-bis-(β-aminoethyl ether)N,N′-tetraacetate (EGTA) and restored by Ca²⁺. Imidazole was without effect on the binding of cyclic AMP to a cyclic AMP-dependent protein kinase from bovine heart. The lack of effect of imidazole on the hydrolysis of physiological levels of cyclic AMP or cyclic GMP suggests that the activity of imidazole to antagonize the effects of various hormones is probably not due to a direct action of imidazole on the hydrolysis of cyclic AMP or cyclic GMP.     

Cyclic nucleotide hydrolysis in the thyroid gland. General properties and key role in the interrelations between concentrations of adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate.

Phosphodiesterase activities of horse (and dog) thyroid soluble fraction were compared with either cyclic AMP (adenosine 3':3'-monophosphate) or cyclic GMP (guanosine 3':5'-monophosphate) as substrate. Optimal activity for cyclic AMP hydrolysis was observed at pH 8, and at pH 7.6 for cyclic GMP. Increasing concentrations of ethyleneglycol bis(2-aminoethyl)-N,N'-tetraacetic acid inhibited both phosphodiesterase activities; in the presence of exogenous Ca2+, this effect was shifted to higher concentrations of the chelator. In a dialysed supernatant preparation, Ca2+ had no significant stimulatory effect, but both Mg2+ and Mn2+ increased cyclic nucleotides breakdown. Mn2+ promoted the hydrolysis of cyclic AMP more effectively than that of cyclic GMP. For both substrates, substrate velocity curves exhibited a two-slope pattern in a Hofstee plot. Cyclic GMP stimulated cyclic AMP hydrolysis, both nucleotides being at micromolar concentrations. Conversely, at no concentration had cyclic AMP any stimulatory effect on cyclic GMP hydrolysis. 1-Methyl-3-isobutylxanthine and theophylline blocked the activation by cyclic GMP of cyclic GMP of cyclic AMP hydrolysis, whereas Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone), a non-methylxanthine inhibitor of phosphodiesterases, did not alter this effect. In dog thyroid slices, carbamoylcholine, which promotes an accumulation of cyclic GMP, inhibits the thyrotropin-induced increase in cyclic AMP. This inhibitory effect of carbamoylcholine was blocked by theophylline and 1-methyl-3-isobutylxanthine, but not by Ro 20-1724. It is suggested that the cholinergic inhibitory effect on cyclic AMP accumulation is mediated by cyclic GMP, through a direct activation of phosphodiesterase activity.     

Inhibition of hepatoma cell growth by analogs of adenosine and cyclic AMP and the influence of enzymes in mammalian sera

The following evidence suggests that inhibition of hepatoma cell (HTC) growth by cyclic nucleotides is an adenosine-like effect that is greatly modified by the type and treatment of serum used in the culture medium and is probably not mediated by cyclic AMP-dependent protein kinase: 1) Heating serum reduces its phosphodiesterase content, thereby slowing metabolism of cyclic AMP and reducing the inhibition of HTC cell growth by cyclic AMP; 2) Using medium that contains phosphodiesterase but lacks adenosine deaminase causes adenosine to accumulate from cyclic AMP and increases the toxicity of cyclic AMP; 3) Uridine or cytidine reverses the growth inhibition caused by adenosine, 5'-AMP or cyclic AMP; 4) adenosine, 5'-AMP and N6-(delta 2-isopentenyl) adenosine are more toxic for HTC cells than is cyclic AMP, and N6,O2-dibutyryl cyclic AMP is not toxic; and 5) N6,O2'-dibutyryl cyclic AMP inhibits growth of Reuber H35 cells, but uridine prevents this inhibition of growth. We conclude that most, if not all, of the inhibitory effects of cyclic AMP and N6,O2'-dibutyryl cyclic AMP on HTc and Reuber H35 hepatoma cell growth are due to the generation of toxic metabolites.     

Seventeen Sxy-Dependent Cyclic AMP Receptor Protein Site-Regulated Genes Are Needed for Natural Transformation in Haemophilus influenzae

Natural competence is the ability of bacteria to actively take up extracellular DNA. This DNA can recombine with the host chromosome, transforming the host cell and altering its genotype. In Haemophilus influenzae, natural competence is induced by energy starvation and the depletion of nucleotide pools. This induces a 26-gene competence regulon (Sxy-dependent cyclic AMP receptor protein [CRP-S] regulon) whose expression is controlled by two regulators, CRP and Sxy. The role of most of the CRP-S genes in DNA uptake and transformation is not known. We have therefore created in-frame deletions of each CRP-S gene and studied their competence phenotypes. All but one gene (ssb) could be deleted. Although none of the remaining CRP-S genes were required for growth in rich medium or survival under starvation conditions, DNA uptake and transformation were abolished or reduced in most of the mutants. Seventeen genes were absolutely required for transformation, with 14 of these genes being specifically required for the assembly and function of the type IV pilus DNA uptake machinery. Only five genes were dispensable for both competence and transformation. This is the first competence regulon for which all genes have been mutationally characterized.     

Redox modulation of the response of NADH oxidase activity of rat liver plasma membranes to cyclic AMP plus ATP

NADH oxidase activity of rat liver plasma membranes was inhibited by lowconcentrations (1-100 nM) of ATP. The inhibition was amplified by additionof nanomolar concentrations (0.1-10) of cyclic AMP. The inhibition wascomplex and related to a marked increase in the Km for NADH at high NADHconcentrations together with a concomitant decrease in the Vmax. In theabsence of added or residual ATP, cyclic AMP was without effect. Theresponse of cyclic AMP + ATP was inhibited by low concentrations of theselective inhibitor of cyclic AMP-dependent protein kinase, H-89 but not bystaurosporin. The Vmax but not the Km was modified by treating the plasmamembranes with a mild oxidizing agent, N-chlorosuccinamide, or with thereducing agent, dithiothreitol. In the presence of dithiothreitol, the Vmaxwas reduced by cyclic AMP + ATP. In contrast, in the presence ofN-chlorosuccinamide, the Vmax was increased by cyclic AMP + ATP relative tocyclic AMP + ATP alone. Thus, the effect of cyclic AMP + ATP on the Vmaxcould be either an increase or a decrease depending on whether the membraneswere oxidized or reduced. The results demonstrate regulation of NADH oxidaseactivity of rat liver plasma membranes through cyclic AMP-mediatedphosphorylation by membrane-located protein kinase activities where thefinal response is dependent on the oxidation-reduction status of the plasmamembranes.     

An assessment of the ability of insulin-stimulated cyclic AMP phosphodiesterase to decrease hepatocyte intracellular cyclic AMP concentrations.

Treatment of hepatocytes with either NH4Cl (10mM) or fructose (10mM) blocks insulin's activation of the 'dense-vesicle' cyclic AMP phosphodiesterase. The ability of insulin (10 nM) to decrease intracellular cyclic AMP concentrations raised by glucagon (10 nM) was unaffected by pre-treatment with either NH4Cl (10 mM) or fructose (10 mM). It is concluded that the 'dense-vesicle' enzyme does not play a significant role in this action of insulin and that as yet unidentified cyclic AMP phosphodiesterase(s) must be activated by insulin. Treatment of hepatocytes with either NH4Cl or fructose appeared to increase, reversibly, cyclic AMP phosphodiesterase activity. When N6-(phenylisopropyl)adenosine was used to prevent glucagon from blocking insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase activity, insulin's ability to decrease intracellular cyclic AMP concentrations in glucagon-treated hepatocytes was increased markedly. Insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase activity can exert a potent effect in decreasing intracellular cyclic AMP concentrations elevated by glucagon.     

Cyclic adenosine monophosphate receptor: effect of cyclic AMP analogues on DNA binding and proteolytic inactivation.

The cyclic AMP receptor protein of Escherichia coli in the presence of cyclic AMP undergoes a conformational change resulting in an increased affinity for DNA and an increased susceptibility to attack by proteolytic enzymes resulting in loss of DNA binding capacity. Of several cyclic nucleotides tested only cyclic AMP and cyclic tubercidin monophosphate are able to effect the conformational transition in cyclic AMP receptor protein, prerequisite to proteolytic inactivation or DNA binding. Other analogues such as cyclic GMP or cyclic IMP which are competitive inhibitors of cyclic AMP do not support DNA binding or proteolytic inactivation.     

Production of cyclic AMP and adenosine by the brain and prothoracic glands of Manduca sexta

Relatively large amounts of cyclic AMP are produced by the prothoracic glands (source of the insect moulting hormone or moulting hormone percursor) of the tobacco hornworm, Manduca sexta. Pharate pupal glands produce more cyclic AMP than early fifth instar larval glands, and the addition of aminophylline enhances cyclic AMP accumulation. The much lower cyclic AMP level in the absence of aminophylline indicates the presence of potent cyclic AMP phosphodiesterase activity. Brains (sources of the prothoracicotropic hormone) also produce cyclic AMP but at a lower rate. Brains efficiently produce adenosine from ATP while β-ecdysone inhibits adenosine formation in early fifth instar larval brains. β-Ecdysone stimulates adenyl cyclase in brains of both stages when aminophylline and fluoride are present but has no effect on cyclic AMP accumulation in prothoracic glands. The absence of fluoride greatly reduces the amount of cyclic AMP produced by prothoracic glands when aminophylline is present. No cyclic AMP is accumulated in prothoracic glands when both fluoride and aminophylline are absent or in brains when fluoride is absent, notwithstanding the presence of aminophylline. Other insect tissues were also analysed for cyclic AMP production and none showed levels nearly as high as the prothoracic glands, suggesting a close relationship between cyclic AMP production and the function of the gland.     

Calcium modulates the generation of inositol 1,3,4-trisphosphate in human platelets by the activation of inositol 1,4,5-trisphosphate 3-kinase.

Histamine (0.5 mM) stimulated the cyclic AMP content of cell suspensions containing greater than 80% parietal cells. Epidermal growth factor (EGF) inhibited this stimulatory effect of histamine, but had no effect on basal cyclic AMP content. The half-maximally effective concentration of EGF for inhibition of histamine-stimulated cyclic AMP was 3.9 nM. The equivalent measurement for the inhibition of histamine-stimulated aminopyrine accumulation was 3.0 nM. Aminopyrine accumulation was measured because it provides an index of the secretory activity of the cell. The cyclic AMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) prevented the inhibitory effect of EGF on cyclic AMP content. This effect of IBMX was not caused by its ability to raise cellular cyclic AMP content in the presence of histamine. Prevention by IBMX of the inhibitory action of EGF on histamine-stimulated aminopyrine accumulation had been shown previously [Shaw, Hatt, Anderson & Hanson (1987) Biochem. J. 244, 699-704]. EGF stimulated prostaglandin E2 (PGE2) production in the cell fraction containing greater than 80% parietal cells, with the half-maximally effective concentration being 7.5 nM. EGF was ineffective in stimulating PGE2 production if the cell fraction was depleted of parietal cells (12%), or if 0.5 mM-histamine was added to the enriched parietal-cell fraction. In conclusion, EGF may inhibit histamine-stimulated acid secretion by decreasing the cyclic AMP content of parietal cells. This effect could be mediated by an increase in cyclic AMP phosphodiesterase activity, but it is unlikely to involve an effect of EGF on parietal-cell prostaglandin production.     

Negative control of TSH action by iodide and acetylcholine: mechanism of action in intact thyroid cells.

Iodide, a substrate of thyroid metabolism, and acetylcholine depress cyclic AMP intracellular content and secretion in dog thyroid slices under TSH stimulation. A direct or indirect pseudocompetitive effect at the level of TSH receptor interaction has been rejected. Iodide and carbachol, both inhibited cyclic AMP accumulation in TSH stimulated dog thyroid slices but only the effect of carbachol was suppressed in the presence of isobutylmethylanthine. Ro 20-1724 did not relieve either inhibitory effect. Carbachol greatly enhanced cyclic AMP disposal in TSH prestimulated slices after the cut off of hormone action by a trypsin treatment. This effect was also suppressed by isobutylmethylxanthine but not by Ro 20-1724. No action of iodide could be evidenced on cyclic AMP disposal in similar slices, although a clear effect after the same time of iodide action was observed on cyclic AMP accumulation. Neither carbachol, nor iodide depresses ATP levels in these slices. The data suggest that carbachol exerts its action through an activation of cyclic AMP disappearance probably by an activation of cyclic AMP phosphodiesterase and that iodide, through an oxidized intermediate, experts its inhibitory effect at the level of cyclic AMP synthesis.     

Dopamine- and octopamine-sensitive adenylate cyclase in the brain of adultCulex pipiens mosquitoes

The effects of dopamine and octopamine on adenylate cyclase activity were studied on the head homogenate of adult Culex pipiens mosquitoes in vitro. Both dopamine and octopamine were shown to increase the cyclic AMP content in the homogenate. The antagonist haloperidol blocked the production of cyclic AMP induced from dopamine but had no effect on the production of cyclic AMP induced by octopamine at the concentrations tested. The opiate agonist etorphine was ineffective at reducing cyclic AMP levels induced by either dopamine or octopamine at the concentrations tested.     

Growth control in chick embryo fibroblasts; no evidence for a specific role for cyclic purine nucleotides

Cyclic AMP and cyclic GMP concentrations were measured in cultures of normal chick embryo fibroblasts and those transformed by Rous sarcoma virus under different growth conditions. No significant and reproducible correlation between the nucleotide levels and the rate of proliferation was observed. Neither release of normal cells from density dependent inhibition of growth nor transformation of the cultures by different strains of Rous sarcoma virus affected the concentrations of cyclic AMP or cyclic GMP. Activities of cellular cyclic nucleotide phosphodiesterases, enzymes involved in regulating the level of the nucleotides, were not directly affected by growth-stimulating concentrations of insulin or neuraminidase. Growth stimulation by insulin did not alter the activities of cellular cAMP-dependent protein kinase. These results do not support the hypothesis that cyclic AMP or cyclic GMP has a specific role in the growth control of chick embryo fibroblasts.