Actions of vasopressin and isoprenaline on the ionic transport across the isolated frog skin in the presence and the absence of adenyl cyclase inhibitors MDL12330A and SQ22536.

1. The effects of both adenyl cyclase inhibitors (MDL12330A and SQ22536) have been studied on the ionic transport induced by vasopressin and isoprenaline across the frog skin. 2. MDL12330A inhibits the vasopressin action on the short-circuit current (SCC), confirming that this effect is cAMP-mediated. 3. On the other hand, isoprenaline action on the SCC is unaffected by MDL12330A. However, this lack of effect is not a sufficient argument against the role of cAMP in this action; in fact, as MDL12330A is also an inhibitor of cAMP phosphodiesterase, this action could mask the inhibitory effect of the drug on adenyl cyclase. 4. By using the other adenyl cyclase inhibitor (SQ22536), probably deprived of effect on the cAMP phosphodiesterase, we obtained a strong inhibition of isoprenaline action on the SCC. Thus we conclude that the actions of isoprenaline on the ionic transport across the frog skin are also cAMP-mediated.     

Metabolism of Adenosine 3′,5′-Cyclic Monophosphate and Induction of Fruiting Bodies in Coprinus macrorhizus

The adenyl cyclase and phosphodiesterase metabolizing adenosine 3',5'-cyclic monophosphate (cyclic AMP) were detected in mycelia of strains of Coprinus macrorhizus which form fruiting bodies, but not in those of strains which do not form fruiting bodies. The adenyl cyclase synthesized cyclic AMP from adenosine triphosphate. The phosphodiesterase degr[UNK]ded cyclic AMP to adenosine-5'-monophosphate and was inhibited by adenosine-3'-monophosphate, theophylline, and caffeine. The strains which form fruiting bodies incorporated and metabolized cyclic AMP, but strains which do not form fruiting bodies did not. The possible participation of cyclic AMP in the induction of fruiting bodies is discussed.     

Developmental pattern of cAMP, adenyl cyclase, and cAMP phosphodiesterase in the palate, lung, and liver of the fetal mouse: alterations resulting from exposure to methylmercury at levels inhibiting palate closure

Exposure to methylmercury (MeHg: 10 mg Hg/kg maternal body weight) on 12(6) (days hours) of gestation significantly delays palate closure in the Swiss Webster CFW mouse. The cAMP content and activity of adenyl cyclase and phosphodiesterase (PDE) were measured in the tissues of control and MeHg-induced cleft palates between 13(6) and 17(6) of gestation. Lung and liver were investigated similarly to determine if MeHg affected the adenyl cyclase system of the palate in a unique manner. In control palatal tissue, cAMP levels increased sharply from 13(22) (undetectable) to 14(6) (maximum). PDE activity increased similarly up to 14(2), but decreased 50% between 14(2) and 14(6). Since it has been reported that cAMP induces the synthesis of PDE, the difference in cAMP/PDE from 13(22) to 14(2) and from 14(2) to 14(6) suggests the localization of relatively high levels of cAMP in at least two separate compartments. Between 14(6) and 14(10), the adenyl cyclase activity of control palates decreased significantly. This rapid decrease suggests relatively high adenyl cyclase activity in the medial edge epithelial cells which undergo autolysis prior to shelf fusion (centered at 14(15). Maternal MeHg administration at 12(6) delayed the median time of palatal shelf rotation (14(13)) by 5 hours, and significantly altered the developmental pattern of the adenyl cyclase system. Thus, the increase in cAMP between 14(2) and 14(6) was abolished and the decrease in adenyl cyclase activity between 14(6) and 14(10) was delayed by almost 20 hours. These changes may be manifestions of a MeHg-induced delay in medial edge epithelial cell differentiation. In a previous study, we observed that the fetal liver exhibits the highest MeHg concentration of all tissues. Since MeHg only slightly altered the adenyl cyclase system of the fetal liver compared to the lung and palate (in which MeHg uptake is considerably less), it may be that the effects of MeHg on palatal tissue are not due to a direct effect of MeHg on components of the adenyl cyclase system.     

Bursicon-induced adenyl cyclase activity in the haemocytes of the American cockroach

Partially purified preparations of bursicon were injected, between ecdyses, into American cockroaches (Periplaneta americana) held at 4°C. After short periods of time, the haemocytes were removed and found to contain appreciable amounts of cyclic adenosine 5′-monophosphate (cAMP). Identical experiments using animals reared at 23°C did not result in the accumulation of cAMP. These and additional data suggest that phosphodiesterase is not active during the initial stages of bursicon-stimulated adenyl cyclase.Incubation of haemocyte soluble proteins in the presence of [³H]cAMP, followed by molecular sieve chromatography on P-60 polyacrylamide gel, shows the binding of cAMP to large molecular weight proteins. Similar experiments, but with phosphodiesterase inhibition revealed a reduced level of radioactive bound protein. From these studies one can assume that the haemocytes contain a soluble cAMP-dependent protein kinase.If either [¹⁴C]-leucine or [¹⁴C]-tyrosine is incubated with whole blood taken from animals between ecdyses, little or no uptake by the haemocytes can be seen. Identical studies, but with the addition of cAMP, results in a dramatic enhancement of amino acid uptake. It appears that bursicon stimulates blood-cell adenyl cyclase, which in turn produces cAMP. The cAMP subsequently activates a specific protein kinase that phosphorylates potential membrane proteins. The membrane phosphoprotein may be instrumental in the enhanced uptake of amino acids.     

The effect of divalent cations on aggregation of Dictyostelium discoideum.

Following the initiation of development, amoebae of Dictyostelium discoideum aggregate chemotactically toward cyclic AMP (cAMP). Adenyl cyclase, cAMP phosphodiesterase, and cAMP binding sites all increase 20--40 fold during the first few hours of development. It has been shown that addition of 1 mM EDTA and 5 mM MgCl2 accelerates the aggregation process. Likewise, the calcium ionophore, A23187, leads to precocious aggregation while 4 X 10(-5) M progesterone considerably delays it. These treatments have now been shown to result in increased accumulation of adenyl cyclase in the case of EDTA and Mg2+ or the ionophore and greatly decreased accumulation in the case of the steroid. Treatment with EDTA and Mg2+ or the ionophore has been shown not only to accelerate aggregation in wild-type amoebae but to overcome complete blocks to aggregation in certain mutant strains. We have found that addition of Mn2+ will also permit aggregation of mutant cells otherwise unable to aggregate. This divalent ion, unlike EDTA and Mg2+ or the ionophore, was shown to directly stimulate adenyl cyclase. Calcium ions were also found to affect the enzyme such that at Ca2+ concentrations found within the cells the great majority of the activity is inhibited. Manganese ions can overcome the inhibition by Ca2+. These findings show that conditions which stimulate aggregation result in increased activity of adenyl cyclase either by increased accumulation of the enzyme or by increased activity of the available enzyme, and support the proposed central role of adenyl cyclase in aggregation.     

ONTOGENETIC DEVELOPMENT OF ADENYL CYCLASE AND PHOSPHODIESTERASE IN RAT BRAIN

Abstract— The activities of adenyl cyclase and phosphodiesterase were determined in homogenates of cerebrum, cerebellum and brain stem of rats of 1 day to 9 weeks of postnatal age. The activity of cerebral and brain stem adenyl cyclase measured either in the absence or presence of sodium fluoride increased rapidly for 2 weeks, reached at 20 days a maximum about three times (brain stem) or six times (cerebrum) that seen on day 1 and then declined slightly during the next several weeks. In contrast, activity of cerebrellar adenyl cyclase increased more slowly and reached a maximum at about 32 days. Activity of phosphodiesterase in cerebrum and brain stem increased several-fold during brain maturation; however, enzymic activity in the cerebellum decreased during the entire 9 week period.
In the pineal gland, adenyl cyclase activity measured in the absence of norepinephrine or sodium fluoride did not change significantly with age. However, enzymic activity measured in the presence of these agents increased with the age of the rat. Bilateral removal of the superior cervical ganglia at 1 day of age is known to arrest the sympathetic innervation of the pineal gland but did not prevent the development of this adenyl cyclase system activated by catecholamines or fluoride.     

Cyclic 3', 5'-adenosine monophosphate phosphodiesterase mutants of Salmonella typhimurium.

Positive selection procedures for mutants of Salmonella typhimurium lacking cyclic 3', 5'7-adenosine monophosphate (cAMP) phosphodiesterase have been devised. The gene (cpd) coding for this enzyme has been located on the chromosome and shown to be 25% co-transducible with metC using phage P22. The mutants have been used to investigate the role of the enzyme in the control of genes whose expression is known to be dependent on cAMP. Significant alterations in the regulation of some but not others of these genes have been observed in these mutants. Mutants lacking the cAMP phosphodiesterase are more sensitive than their parents to a variety of antibiotics that appear to enter the cell through cAMP-dependent transport systems. They grow faster than the wild type on succinate-ammonia-salts, and glucose-proline-salts media and are inhibited by added cAMP on glucose, citrate, or glycerol-ammonia salts media whereas the wild type is unaffected. Neither the growth of Salmonella typhimurium on glycerol or citrate media nor the level of acid hexose phosphatase in the strain is affected by the loss of cAMP phosphodiesterase. In addition, the mutant strains are extremely sensitive to high levels of cAMP. Loss of the cAMP phosphodiesterase in strains unable to synthesize cAMP (adenyl cyclase negative) reduces by 10-fold the requirement for exogenous cAMP for expression of catabolite-sensitive phenotypes. These results suggest that through its control of cAMP levels in the cell the phosphodiesterase may be involved in the regulation of certain classes of catabolite-sensitive operaons and also in protecting the cell against high levels of cAMP.     

The Effect of Divalent Cations on Aggregation of Dictyostelium discoideum

Following the initiation of development, amoebae of Dictyostelium discoideum aggregate chemotactically toward cyclic AMP (cAMP). Adenyl cyclase, cAMP phosphodiesterase, and cAMP binding sites all increase 20–40 fold during the first few hours of development. It has been shown that addition of 1 mM EDTA and 5 mM MgCl₂ accelerates the aggregation process. Likewise, the calcium ionophore, A23187, leads to precocious aggregation while 4 × 10⁻⁵ M progesterone considerably delays it These treatments have now been shown to result in increased accumulation of adenyl cyclase in the case of EDTA and Mg²⁺ or the ionophore and greatly decreased accumulation in the case of the steroid.
Treatment with EDTA and Mg²⁺ or the ionophore has been shown not only to accelerate aggregation in wild-type amoebae but to overcome complete blocks to aggregation in certain mutant strains. We have found that addition of Mn²⁺ will also permit aggregation of mutant cells otherwise unable to aggregate. This divalent ion, unlike EDTA and Mg²⁺ or the ionophore, was shown to directly stimulate adenyl cyclase. Calcium ions were also found to affect the enzyme such that at Ca²⁺ concentrations found within the cells the great majority of the activity is inhibited. Manganese ions can overcome the inhibition by Ca²⁺.
These findings show that conditions which stimulate aggregation result in increased activity of adenyl cyclase either by increased accumulation of the enzyme or by increased activity of the available enzyme, and support the proposed central role of adenyl cyclase in aggregation.     

The relationship of phosphodiesterase to the developmental cycle of Dictyostelium discoideum.

Determination of the rate of total phosphodiesterase production by Dictyostelium discoideum shows that a dramatic rise in enzyme production occurs after 3 hours of cell starvation. Use of imposed cAMP pulses indicate that this increase is related to the developmental program of the amoebae and is probably due to a stimulation of adenyl cyclase.     

Adenyl cyclase in the haemocytes of the American cockroach

Incubation of ¹⁴C-adenosinetriphosphate with lysed haemocytes produces ¹⁴C-cyclic 3′,5′-adenosinemonophosphate (cAMP). The addition of phosphodiesterase to similar reaction mixtures results in the conversion of cAMP to 5′-adenosinemonophosphate.Differential centrifugation of the haemocyte preparation revealed that adenyl cyclase activity occurred in both membrane bound and soluble fractions, although the evidence suggests that the enzyme activity was originally particulate.The significance of these data is discussed in regard to the sclerotization process and the mode of action of bursicon.     

Changes in intracellular cAMP level and activities of adenylcyclase and phosphodiesterase during meiosis of lily microsporocytes.

In the yeasts, Saccharomyces cerevisiae and Schyzosaccharomyces pombe, reduction of intracellular cyclic adenosine monophosphate (cAMP) is known to trigger the sporulation processes by activating various meiosis specific genes. In order to ascertain whether a similar mechanism is operative in higher plants, we carried out preliminary studies on lily microsporocytes. Measurement of cAMP levels as well as the activities of adenyl cyclase and phosphodiesterase in somatic cells and different stages of meiosis, and arrest of its in protoplasts cultured under conditions of high cAMP provided direct evidence that similar phenomena occur in plant meiocytes as earlier documented in yeasts.     

Regulation of multiple tip formation by caffeine in cellular slime molds

ABSTRACT: BACKGROUND: The multicellular slug in Dictyostelium has a single tip that acts as an organising centre patterning the rest of the slug. High adenosine levels at the tip are believed to be responsible for this tip dominance and the adenosine antagonist, caffeine overrides this dominance promoting multiple tip formation. RESULTS: Caffeine induced multiple tip effect is conserved in all the Dictyostelids tested. Two key components of cAMP relay namely, cAMP phosphodiesterase (Pde4) and adenyl cyclase-A (AcaA) levels get reduced during secondary tip formation in Dictyostelium discoideum. Pharmacological inhibition of cAMP phosphodiesterase also resulted in multiple tips. Caffeine reduces cAMP levels by 16.4, 2.34, 4.71 and 6.30 folds, respectively in D. discoideum, D. aureostipes, D. minutum and Polysphondylium pallidum. We propose that altered cAMP levels, perturbed cAMP gradient and impaired signalling may be the critical factors for the origin of multiple tips in other Dictyostelids as well. In the presence of caffeine, slug cell movement gets impaired and restricted. The cell type specific markers, ecmA (prestalk) and pspA (prespore) cells are not equally contributing during additional tip formation. During additional tip emergence, prespore cells transdifferentiate to compensate the loss of prestalk cells. CONCLUSION: Caffeine decreases adenyl cyclase--A (AcaA) levels and as a consequence low cAMP is synthesised altering the gradient. Further if cAMP phosphodiesterase (Pde4) levels go down in the presence of caffeine, the cAMP gradient breaks down. When there is no cAMP gradient, directional movement is inhibited and might favour re-differentiation of prespore to prestalk cells.     

Hemicyst formation stimulated by cyclic AMP in dog kidney cell line MDCK.

Certain epithelial cell lines have morphologic, physiologic, biochemical and pharmacologic characteristics of transporting epithelia from intact organs. In this paper we show that dibutyryl cyclic AMP, 5' AMP, adenosine and cyclic AMP phosphodiesterase inhibitors stimulate hemicyst formation by the dog kidney cell line MDCK. It is suggested that this effect is explained by elevation of intracellular cyclic AMP levels by means of an exogenous non-metabolizable source of cyclic AMP, phosphodiesterase inhibition or adenyl cyclase stimulation. Since hemicyst formation is in part due to transepithelial fluid transport, these findings raise the possibility that this fraction might be modulated by cAMP in an established cell line. We believe that cultured epithelial cells may provide an exploitable model system to investigate at the cellular and subcellular levels, the mechanism by which cyclic AMP modifies water and solute movements across epithelia.     

Which postsynaptic action of dopamine is mediated by cyclic AMP?

The experimental basis, for proposals that adenosine 3' ,5'-cyclic monophosphate (cAMP) acts as intracellular mediator of one or the other postsynaptic actions of dopamine (DA) in mammalian sympathetic ganglia, is analyzed. These synaptic actions of DA are (I) a hyperpolarizing one, as the direct transmitter for the slow-inhibitory postsynaptic potential (s-IPSP); and (II) a modulatory one, inducing an enduring enhancement of the slow-excitatory postsynaptic response (s-EPSP) to another transmitter, acetylcholine (ACh).(A) Stimulation of adenyl cyclase by DA or appropriate neural input appears generally to support either role; however, the comparative characteristics of DA and norepinephrine (NE) in relation to adenyl cyclase do not appear to be in accord with those in relation to hyperpolarizing actions. (B) Postsynaptic actions of cAMP do not support a role in DA action-I, but they are fully appropriate for DA action-II. (C) Phosphodiesterase inhibition by the relatively potent and selective agent RO-20-1724, under conditions reported to protect and increase cAMP in these ganglia, is shown not to augment the s-IPSP or DA-hyperpolarization; although theophylline does augment these responses, this effect is shown not to be attributable to an inhibition of phosphodiesterase, and it does not provide support for a role in DA action-I. Effects of these inhibitors are at least compatible with the proposed modulatory role-II for cAMP. (D) The timing of the putative chemical reactions involved in a mediation by cAMP would appear to be far too slow for the purposes of DA action-I (s-IPSP response), but it is readily accommodated by the slow onset and development of the modulatory change induced by DA action-II.The suggestion that guanosine 3', 5'-cyclic monophosphate (cGMP) may mediate the slow muscarinic depolarizing response to ACh (s-EPSP) has gained definitive experimental support. Suitable cholinergic stimulation of guanyl cyclase has been demonstrated. The postsynaptic action of cGMP in a low concentration range fits with the unique characteristics of the s-EPSP, at least for cells with normal, not already depolarized, resting membrane potentials. cGMP has also been found capable of antagonizing the modulatory action of either DA (action-II) or of cAMP, but only in a remarkably time-dependent manner.It is concluded that cAMP does not mediate the inhibitory synaptic DA action-I (s-IPSP response), but that it probably does mediate the enduring modulatory change in the s-EPSP (DA action-II). cGMP probably does mediate the production of the s-EPSP by ACh. cAMP would thus have a synergistic, rather than opposing, physiological action in relation to cGMP. A re-examination of the functional significance of related DA-activated adenyl cyclase systems in the brain is suggested.     

Changes of the activities of adenyl cyclase and cAMP-phosphodiesterase and of the level of 3'5' cyclic adenosine monophosphate in rat mammary gland during pregnancy and lactation

The activities of adenyl cyclase, the low- and high-K_m cAMP phosphodiesterases and the tissue level of cAMP have been measured in mammary glands taken from pregnant and lactating rats. Adenyl cyclase reaches its maximum activity late in pregnancy and thereafter falls continuously throughout lactation. The high-K_m phosphodiesterase rises slowly in pregnancy but reaches a maximum late in lactation. The tissue cAMP content is maximal at the end of pregnancy and then falls progressively to its lowest level by the sixteenth day of lactation. These changes are discussed in relation to the metabolic adaptation of the gland and its responsiveness to hormonal stimulation.     

Relationship between the tonus of the walls of the internal carotid artery and cyclic adenylmonophosphate

Experiments were carried out in dogs with the use of resistography of circulatory-isolated internal carotid artery. Both activation of adenyl cyclase by adenosine and inhibition of phosphodiesterase by theophylline in the vascular wall result in a significant decrease in the vasoconstrictor effect of prestaglandin E2. On the contrary, inhibition of adenyl cyclase by the beta-blocker propranolol or activation of phosphodiesterase by imidazol in the vascular wall causes potentiation of vasoconstriction brought about by prostaglandin E2. Thus the endogenous cyclic AMP either antagonizes the vasoconstrictor effect of prostaglandin E2, or the latter affects vascular smooth muscles by influencing the activity of enzymes that are responsible for the cyclic AMP level in the vascular wall.     

An assay for adenyl cyclase based on a combination of sodium borate electrophoresis and paper chromatography.

We describe a method for the assay of adenyl cyclase in whole tissue homogenates. Adenosine 3′:5′-cyclic monophosphate (cAMP) formed from α-³²P-, ¹⁴C- or ³H-labeled adenosine 5′-triphosphate (ATP) substrate is isolated from all known ATP metabolites and an unknown metabolite by electrophoresis in 1% sodium borate for 40 min, followed by overnight descending chromatography in 95% ethanol:1 m ammonium acetate (70:30). The purity of the cAMP isolated is established by chromatographic techniques as well as by utilizing a purified cyclic nucleotide phosphodiesterase. The method described here also makes possible the measurement of phosphodiesterase activity in homogenates. It is rapid enough to allow routine assay of 180 samples per day, although the number of samples processed depends on the number of electrophoretic and chromatographic units available.     

Properties of two cyclic nucleotide-deficient mutants of Neurospora crassa.

Studies on the crisp-1 (cr-1), cyclic adenosine 3',5'-monophosphate (cAMP)-deficient mutants of Neurospora crassa were undertaken to characterize the response of these mutants to exogenous cyclic nucleotides and cyclic nucleotide analogs. A growth tube bioassay and a radioimmune assay for cyclic nucleotides yielded the following results. (i) 8-Bromo cAMP and N6-monobutyryl cAMP but not dibutyryl cAMP are efficient cAMP analogs in Neurospora, stimulating mycelial elongation of the cr-1 mutants. Exogenous cyclic guanosine 3'5'-monophosphate (cGMP) also stimulates such mycelial elongation. (ii) Both cAMP levels and cGMP levels found in cr-1 mycelia are lower than those in wild type. However, the levels of both cyclic nucleotides are normal in conidia of cr-1. The data on cr-1 mycelia and those reported earlier in Escherichia coli (M. Shibuya, Y. Takebe, and Y. Kaziro (Cell 12:528-528, 1977) show a previously unexpected relationship between cAMP and cGMP metabolism in microorganisms. The semicolonial morphology of another adenylate cyclase-deficient mutant of Neurospora, frost, was not corrected by exogenous cyclic nucleotides or by phosphodiesterase inhibitors indicating that the frost morphology is probably not caused by low endogenous cAMP levels. The low adenylate cyclase activity and the abnormal morphology of frost may be related separately to the linolenate deficiency reported in the mutant.     

Mechanism of hormonally induced refractoriness of ovarian adenylate cyclase to luteinizing hormone and prostaglandin E.

Stimulation by prostaglandinE2 (PGE2) or luteinizing hormone (LH) of cyclic AMP (cAMP) production by rat Graafian follicles was reduced when the follicles were cultured for 3-6 hours in PGE2 or 12-24 hours in cAMP. The follicles regained adenylcyclase response to PGE2 when held in a PG-free medium, but refractoriness to LH remained even after culture without LH for 8 hours or in anti-LH antiserum. Follicle desensitization to LH was not associated by a decrease in total number of LH-binding sites, nor by an altered activity of cAMP phosphodiesterase. Desensitized follicles responded fully to NaF, quanosine triphosphate (GTP), or guanylimidodiphosphate (Gpp(NH)p). Actinomycin D or cycloheximide prevented the development of refractoriness to PGE2 when added with PGE2. Actinomycin D also prevented desentization to LH. Therefore desensitization may involve synthesis of a protein that couples hormone reception to adenyl cyclase.     

Increase in Endogenous and Exogenous Cyclic AMP Levels Inhibits Sclerotial Development in Sclerotinia sclerotiorum

Growth and development of a wild-type Sclerotinia sclerotiorum isolate were examined in the presence of various pharmacological compounds to investigate signal transduction pathways that influence the development of sclerotia. Compounds known to increase endogenous cyclic AMP (cAMP) levels in other organisms by inhibiting phosphodiesterase activity (caffeine and 3-isobutyl-1-methyl xanthine) or by activating adenylate cyclase (NaF) reduced or eliminated sclerotial development in S. sclerotiorum. Growth in the presence of 5 mM caffeine correlated with increased levels of endogenous cAMP in mycelia. In addition, incorporation of cAMP into the growth medium decreased or eliminated the production of sclerotia in a concentration-dependent manner and increased the accumulation of oxalic acid. Inhibition of sclerotial development was cAMP specific, as exogenous cyclic GMP, AMP, and ATP did not influence sclerotial development. Transfer of developing cultures to cAMP-containing medium at successive time points demonstrated that cAMP inhibits development prior to or during sclerotial initiation. Together, these results indicate that cAMP plays a role in the early transition between mycelial growth and sclerotial development.