A Novel Effect of Cyclic AMP on Capacitative Ca2+ Entry in Cultured Rat Cerebellar Astrocytes

One of the most important intracellular Ca2+ regulatory mechanisms in nonexcitable cells, "capacitative Ca2+ entry" (CCE), has not been adequately studied in astrocytes. We therefore investigated whether CCE exists in cultured rat cerebellar astrocytes and studied the roles of cyclic AMP (cAMP) and protein kinase C (PKC) in CCE. We found that (1) at least two different intracellular Ca2+ stores, the endoplasmic reticulum and mitochondria, are present in cerebellar astrocytes; (2) CCE does exist in these cells and can be inhibited by Ni2+, miconazole, and SKF 96365; (3) CCE can be directly enhanced by an increase in intracellular cAMP, as 8-bromoadenosine 3',5'-cyclic monophosphate (8-brcAMP), forskolin, and isobutylmethylxanthine have stimulatory effects on CCE; and (4) neither of the two potent protein kinase A (PKA) inhibitors, H8 and H89, nor a specific PKA agonist, Sp-adenosine 3',5'-cyclic monophosphothioate, had a significant effect on cAMP-enhanced Ca2+ entry. The [Ca2+]i increase was not due to a release from calcium stores, hyperpolarization of the membrane potential, inhibition of calcium extrusion, or a change in pHi, suggesting that cAMP itself probably acts as a novel messenger to modulate CCE. We also conclude that activation of PKC results in an increase in CCE. cAMP and PKC seem to modulate CCE by different pathways.     

Stimulation of cyclic AMP in chondrocyte cultures: effects on sulfated-proteoglycan synthesis

The effects of N6,O2'-dibutyryladenosine 3':5'-cyclic monophosphate (DBcAMP), 8-bromoadenosine 3':5'-cyclic monophosphate (8Br-cAMP), 3':5'-cyclic monophosphate (cAMP), L-isoproterenol and L-epinephrine on sulfated-proteoglycan synthesis by rabbit articular chondrocytes were compared. DBcAMP and 8Br-cAMP in the presence or absence of 3-isobutyl-1-methylxanthine (IBMX) stimulated sulfated-proteoglycan biosynthesis after 20 h of incubation. cAMP had no significant effect. Both DBcAMP and 8Br-cAMP increased the hydrodynamic size of the newly synthesized proteoglycan monomer (A1D1) relative to control cultures. By contrast, although isoproterenol and epinephrine stimulated total cAMP synthesis, neither stimulated sulfated-proteoglycan synthesis. Whereas intracellular cAMP accumulated after incubation with DBcAMP and 8Br-cAMP, this was not the case with isoproterenol whether IBMX was present or not. Thus, stimulation of sulfated-proteoglycan synthesis by cAMP analogues in chondrocyte cultures appears to be dependent on increased intracellular cAMP accumulation rather than total cAMP biosynthesis.     

Independent regulation of pyruvate kinase expression by cyclic AMP and prostaglandin F2 alpha in mouse mastocytoma cells

P-815 mouse mastocytoma cells express the K isozyme of pyruvate kinase and the specific activity of this enzyme is increased in response to N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate, 8-bromoadenosine 3':5'-cyclic monophosphate, cholera toxin, and epinephrine, all of which also elevate the intracellular concentration of adenosine 3':5'-cyclic monophosphate. Prostaglandin F2 alpha also increases the cellular activity of this enzyme, but does not increase the adenosine 3':5'-cyclic monophosphate levels. Under all these conditions, the increase in enzymatic activity is accompanied by an equivalent increase in the pyruvate kinase protein level. However, neither the rate of enzyme synthesis nor the level of pyruvate kinase mRNA is elevated by N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate. On the other hand, it does increase the enzyme's half-life. In contrast, prostaglandin F2 alpha increases the rate of synthesis and the level of pyruvate kinase K mRNA, but has no influence on the rate of degradation. Therefore, these cells have two mechanisms which increase pyruvate kinase K levels. One operates via an increase in cAMP level and results in a decrease in the rate of degradation, whereas the other minimizes an upsurge in cAMP levels but still increases pyruvate kinase K activity by increasing its rate of synthesis.     

Differences and similarities between guanosine 3',5'-cyclic monophosphate phosphodiesterase and adenosine 3',5'-cyclic monophosphate phosphodiesterase activities in neuroblastoma cells in culture.

There are phosphodiesterase activities in both particulate and supernatant fractions which hydrolyze guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) with an apparent Km of 2-8 muM and with an apparent Km of 44-222 muM. 4-(3-Butoxy-4-methoxybenzyl-2-imidazolidinone (RO20-1724) did not inhibit cGMP phosphodiesterase activity in homogenates of mouse neuroblastoma cells, but markedly inhibited cAMP phosphodiesterase activity. Papaverine and theophylline inhibited both cGMP and cAMP phosphodiesterase activities to about the same extent. The former was more potent than the latter. The specific activity of cGMP phosphodiesterase as a function of protein concentrations first increased and then decreased. The specific activity of cAMP phosphodiesterase decreased under a similar experimental condition.     

Direct regulation of smooth muscle contractile elements by second messengers

The effects of adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP) and phorbol 12,13 dibutyrate (PDBu) on the Ca2+ sensitivity of the contractile elements in the rat mesenteric artery were investigated, using a method of permeabilizing smooth muscle with Staphylococcal alpha-toxin. Both cAMP and cGMP relaxed the permeabilized rat mesenteric artery at the intracellular Ca2+ concentrations [( Ca2+]i) held constant with Ca2+ EGTA buffer and Ca2+ ionophore, ionomycin. In addition, forskolin and sodium nitroprusside which activate adenylate and guanylate cyclases, respectively, also induced relaxation at a fixed [Ca2+]i. In contrast PDBu which stimulates protein kinase C caused an increase in force at a constant [Ca2+]i which could be partially reversed by cAMP or cGMP. These results indicate that second messengers exert direct control over smooth muscle Ca2+ sensitivity of the contractile elements, which is of physiologic and pharmacologic importance.     

Effects of oxytocin and methacholine on cyclic nucleotide levels of rabbit myometrium

The effects of oxytocin and methacholine on cyclic nucleotide levels in estrogen-primed rabbit myometrium were studied in the presence and absence of 1-methyl-3-isobutyl xanthine (MIX), a phosphodiesterase inhibitor. In the absence of MIX, methacholine increased guanosine 3',5'-cyclic monophosphate (cGMP) levels at a time when contraction was decreasing, but had no influence on adenosine 3',5'-cyclic monophosphate (cAMP) levels. In contrast, oxytocin did not elevate cGMP, but rapidly decreased cAMP levels. MIX (1 mM) increased both cAMP and cGMP levels. Oxytocin or methacholine further increased cGMP, indicating activation of guanylate cyclase. Oxytocin- but not methacholine-induced stimulation of guanylate cyclase was abolished in Ca2+-free solution. Oxytocin increased cAMP over the levels produced by MIX alone, whereas methacholine decreased cAMP below the MIX control values; these effects were insensitive to indomethacin. Tissue levels of cGMP and cAMP did not directly correlate with isometric tension. The results also indicate that both oxytocin and methacholine stimulate guanylate cyclase but have opposing effects on adenylate cyclase of rabbit myometrium.     

Control mechanisms governing the infectivity of Chlamydia trachomatis for hela cells: modulation by cyclic nucleotides, prostaglandins and calcium

Chlamydia trachomatis causes common infections of the eyes and genital tract in man. The mechanism by which this obligate intracellular bacterium is taken into epithelial cells is unclear. The results described here support the concept that chlamydial infections of HeLa cells is under bidirectional cyclic nucleotide control, with guanosine 3':5'-cyclic monophosphate (cGMP) acting as a stimulator, and adenosine 3':5'-cyclic monophosphate (cAMP) as an inhibitor. Treatment of the HeLa cells with the divalent cation ionophore A23187, with carbamoylcholine, or with prostaglandins known to increase the concentration of endogenous cGMP, also increased host cell susceptibility to chlamydial infection. Cyclic GMP was only effective if added at or before chlamydial inoculation, suggesting that its main effect was on chlamydial uptake. The stimulatory effect of cGMP, but nt antagonism, by cAMP, was abolished if the cells were first treated with any of four different inhibitors of prostaglandin synthesis, suggesting a critical role for endogenous prostaglandin synthesis. Centrifugation of chlamydiae on to host cells was followed by a rapid increase in the mobility of Ca2+ across the cell membrane. The interrelationships of these observations and the possibility that chlamydiae and other intracellular pathogens might evoke alterations in host cell prostaglandin and cyclic nucleotide concentrations to aid their own uptake are discussed.     

Cytidylate cyclase: development of assay and determination of kinetic properties of a cytidine 3'',5''-cyclic monophosphate-synthesizing enzyme.

A method is described for the separation of cytidine 3',5'-cyclic monophosphate (cyclic CMP) from cytidine tri-, di- and mono-phosphates and from cytidine 3',5'-cyclic pyrophosphate, cytidine 2'-monophosphate-3',5'-cyclic monophosphate, cytidine 2'-O-aspartyl-3',5'-cyclic monophosphate and cytidine monophosphate, compounds previously shown to be the result of putative cytidylate cyclase activity. This separation, involving elution of a novel bilayer column of QAE-Sephadex and alumina with 0.03 M-HCl, has been incorporated into an assay protocol to determine the enzyme-catalysed conversion of radiolabelled CTP to cyclic CMP. By this assay, cytidylate cyclase activity has been shown to be present in rat lung, spleen, ovary, testes, brain, stomach, liver, heart and kidney preparations; the activity was of a similar order in each tissue and had a sharp pH optimum of 7.0-7.5. The liver preparation had a Vmax. of 1.2 nmol of cyclic CMP formed/min per mg, and a Km of 220 microM-CTP, and although active in the absence of added cations, it was stimulated by Fe2+ and Mn2+ ions. In several of the tissues examined, the cytidylate cyclase activity was inversely proportional to age of the animals.     

Determination of the rates of synthesis and degradation of adenosine 3',5'-cyclic monophosphate in Escherichia coli CRP- and CRP+ strains.

We have developed a method for estimating the rates of synthesis and degradation of adenosine 3',5'-cyclic monophosphate (cAMP) in Escherichia coli during balanced growth. Applying this method, we have found that an E. coli CRP- mutant 5333 (deficient for cAMP receptor protein) synthesizes cAMP about 25 times faster than does its CRP+ parent 1100. This accounts for the abnormally high intracellular and extracellular cAMP accumulation in 5333.     

Inhibition of DNA synthesis of adult rat hepatocytes in primary culture by dibutyrylcytidine 3', 5'-cyclic monophosphate

The effect of dibutyrylcytidine 3',5'-cyclic monophosphate (Bt2cCMP) on DNA synthesis of adult rat hepatocytes in primary culture was examined. Bt2cCMP caused dose-dependent inhibition of the DNA syntheses stimulated by various growth factors including human hepatocyte growth factor (hHGF). Dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) inhibited the DNA synthesis more effectively than Bt2cCMP, but dibutyrylguanosine 3',5'-cyclic monophosphate (Bt2cGMP) and n-butyrate had a slight or null inhibitory effect. When added at the onset of DNA synthesis, Bt2cAMP was much less effective, but Bt2cCMP was still effective. Thus Bt2cCMP is able to inhibit growth factor-stimulated hepatocyte proliferation.     

Cationic membrane conductances induced by intracellularly elevated cAMP and Ca2+: measurements with ion-selective microelectrodes

Adenosine 3',5'-cyclic monophosphate (cAMP) and CaCl2 were injected by a fast and quantitative pressure injection technique into voltage-clamped, identified Helix neurons. Intracellular elevation of cAMP as well as of Ca2+ activated an inward current (IcAMP and IN). To identify the ionic fluxes during IcAMP and IN changes in [Na+]i, [K+]o, [H+]i, and [Cl-]i were measured with ion-selective microelectrodes (ISMs). Near resting potential, Na+ was the main carrier of IcAMP. K+, and less effectively Ca2+, could substitute for Na+ in carrying IcAMP. H+ and Cl- were excluded as current carriers for IcAMP by means of ISMs. Simultaneous to this action, cAMP decreased a K+ conductance. This decrease was associated with a reduction of the K+ efflux activated by long-lasting depolarizing voltage steps, as directly measured with ISMs located near the external membrane surface. The nearly compensatory increase and decrease of two membrane conductances in the same neuron left the cell input resistance unchanged despite the considerable depolarizing action of intracellularly elevated cAMP. IN was also of nonspecific nature. However, our findings indicate less selectivity for the Ca2+-activated nonspecific channels. Large cations such as choline, TEA, and Tris passed nearly as well as Na+ through the channels. Measurements with ISMs showed that [H+]i and [Cl-]i were unchanged during IN. IN was largest in bursting pacemaker neurons compared with other cells of similar size. It was found to be essential for the burst production in these cells. IcAMP, on the other hand, might be involved in the presynaptic facilitatory action of cAMP, which as yet was attributed solely to a reduction of a K+ conductance.     

Identification of Adenosine-3′,5′-Monophosphate as the Bacterial Attractant for Myxamoebae of Dictyostelium discoideum

Adenosine-3',5'-cyclic monophosphate was shown to be the compound found in Escherichia coli responsible for the attraction of the amoebae of the cellular slime mold Dictyostelium discoideum. A number of other nucleotides were tested and the following were active: tubercidin-3',5'-cyclic monophosphate, N(6)-2'-O-dibutyryl-adenosine-3',5'-cyclic monophosphate, 5'-methylene adenosine-3',5'-cyclic monophosphonate, guanosine-3',5'-cyclic monophosphate, uridine-3',5'-cyclic monophosphate, cytidine-3',5'-cyclic monophosphate, inosine-3',5'-cyclic monophosphate, and thymidine-3',5'-cyclic monophosphate. They were less active than adenosine-3',5'-cyclic monophosphate. It is suggested that cyclic adenosine monophosphate secreted by the bacteria is used by the amoebae as a means of sensing and orienting towards food.     

Mapping ligand interactions with the hyperpolarization activated cyclic nucleotide modulated (HCN) ion channel binding domain using a soluble construct

Hyperpolarization activated cyclic nucleotide modulated (HCN) ion channel currents are activated by hyperpolarization and modulated in response to changes in cytosolic adenosine 3',5'-cyclic monophosphate (cAMP) concentrations. A cDNA chimera combining the rat HCN2 cyclic nucleotide binding domain and the DNA binding domain of the cAMP receptor protein (CRP) from E. coli and the histidine tag (HCN2/CRP) was expressed and purified. The construct is capable of forming only non-ligand dependent dimers because the C-linker region of the channel is not present in this construct. The construct binds 8-[[2-[(fluoresceinylthioureido) amino] ethyl] thio] adenosine-3',5'-cyclic monophosphate (8-fluo cAMP) with a Kd of 0.299 microM as determined with a monomer binding model. The Ki values of 20 ligands related to cAMP were measured in order to determine the properties necessary for a ligand to bind to the HCN2 binding domain. This is the first report of cAMP and gunaosine 3',5'-cyclic monophosphate (cGMP) affinities to the HCN2 binding domain being equivalent, even though they modulate the channel with a 10-fold difference in K0.5. Furthermore, the array of ligands measured allows the preference rank order for each purine ring position to be determined: position 1, H > NH2 > O; position 2, NH2 > Cl > H > O; position 6, NH2 > Cl > H > O; and position 8, NH2 > Cl > H > O. Finally, the ability of HCN2/CRP to bind cyclic nucleotide pyrimidine rings at concentrations approximately 1.33 times greater than cAMP suggests that ribofuranose is key for binding.     

The role of intracellular cAMP in renal gluconeogenesis in view of differential action of various cAMP analogues

Effects of various cAMP analogues on gluconeogenesis in isolated rabbit kidney tubules have been investigated. In contrast to N(6),2'-O-dibutyryladenosine-3',5'-cyclic monophosphate (db-cAMP) and cAMP, which accelerate renal gluconeogenesis, 8-bromoadenosine-3',5'-cyclic monophosphate (Br-cAMP) and 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP) inhibit glucose production. Stimulatory action of cAMP and db-cAMP may be evoked by butyrate and purinergic agonists generated during their extracellular and intracellular metabolism resulting in an increase in flux through fructose-1,6-bisphosphatase and in consequence acceleration of the rate of glucose formation. On the contrary, Br-cAMP is poorly metabolized in renal tubules and induces a fall of flux through glyceraldehyde-3-phosphate dehydrogenase. The contribution of putative extracellular cAMP receptors to the inhibitory Br-cAMP action is doubtful in view of a decline of glucose formation in renal tubules grown in the primary culture supplemented with forskolin. The presented data indicate that in contrast to hepatocytes, in kidney-cortex tubules an increased intracellular cAMP level results in an inhibition of glucose production.     

Dehydration partitioned within core protoplast accounts for heat resistance of bacterial spores

Abstract In Escherichia coli , adenosine 3',5'-cyclic monophosphate (cAMP) is excreted into the growth media. Making use of a phosphodiesterase as scavenger of extracellular cAMP we show that: (i) extracellular cAMP does not interfere with cellular functions; (ii) transient accumulation of cAMP, followed by its rapid excretion, elicits a severe repression of catabolic enzymes.     

The micronucleus test and erythropoiesis: effects of cyclic adenosine monophosphate (cAMP) on micronucleus formation

The aim of this study was to determine the mechanism of the rodent bone marrow micronucleus test in relation to erythropoiesis. We have previously reported that an acceleration of erythropoiesis increases the frequency of micronucleated polychromatic erythrocytes (MPCE) induced by mutagens. The blood plasma erythropoietin level increased after the injection of N6-2-O-dibutyladenosine-3',5'-cyclic monophosphate into adenosine 3',5'-cyclic monophosphate (cAMP) at a dose of 500 mg/kg. A peak of erythropoietin induction was observed 3 h after the injection of cAMP. cAMP itself did not induce any micronuclei in erythroblasts of BALB/c mice. So, the frequency of MPCE did not increase after injection of cAMP. The highest frequency of MPCE and the dose-response relationship between the cAMP doses and micronucleus frequency were observed 30 h after injection of mitomycin C (MMC) in mice which had been administered cAMP 24 h previously. The highest effect of cAMP on the increase of MPCE was observed when cAMP was given 24 h before MMC injection, thus indicating that accelerating the multiplication of erythroblasts increases the frequency of MPCE induced by mutagens. The induction of MPCE in the bone marrow by three other chemicals (carboquone, 5-fluorouracil, and vincristine) also increased after pretreatment with cAMP. Our results suggest that the increase of MPCE induced by mutagens can be amplified following the acceleration of erythropoiesis by pretreatment with cAMP.     

Exogenous cAMP and cGMP modulate branching in fusarium graminearum.

A study was made of the effects of adenosine 3',5'-cyclic monophosphate (cAMP), guanosine 3',5'-cyclic monophosphate (cGMP) and choline on the morphology and growth of a wild-type strain (A 3/5) and a highly branched, 'colonial' mutant strain (C106) of Fusarium graminearum. Addition of up to 50 mM-cAMP or cGMP to the medium had no effect on the specific growth rate of strain A 3/5. For strain A 3/5, but not for strain C106, exogenous cAMP caused significant decreases in both mean hyphal extension rate (E) and hyphal growth unit length (G), i.e. cAMP caused mycelia of strain A 3/5 to branch profusely. By contrast, for both strains, cGMP caused significant increases in both E and G, i.e. exogenous cGMP caused mycelia to branch more sparsely. The effects of exogenous cGMP and choline in increasing E and G were synergistic, but the effects of cGMP and choline counteracted the effect of cAMP. The mutant phenotype of strain C106 was not correlated with altered levels of endogenous cAMP or cGMP.     

Photoaffinity labeling of adenosine 3',5'-cyclic monophosphate binding sites of human red cell membranes.

To identify and investigate the cAMP binding sites of human red cell membranes a photoaffinity analog of cAMP, 8-azidoadenosine 3',5'-cyclic monophosphate (8-N3cAMP), has been synthesized. This analog activates cAMP-dependent protein kinase(s) in the red cell membrane. It exhibits tight, but reversible binding to the membranes which is competitive with cAMP. Photolysis of [32P]-8-N3cAMP with red cell membranes results in covalent incorporation of radioactive label onto two specific membrane proteins. This incorporation requires activating light and is reduced to background levels with addition of low levels of cAMP. Prephotolysis of 8-N3cAMP completely abolished its ability to photolabel membrane proteins. Both the reversible and photocatalyzed binding of 8-N3cAMP show saturation kinetics. The molecular weights of the two primarily labeled proteins are approximately 49,000 and 55,000. The differential effects of cAMP, ATP, and adenosine on the photocatalyzed incorporation of [32P]-8-N3cAMP onto these two proteins suggest that they have biochemically different properties. The potential usefulness of this compound for investigating various molecular aspects of cAMP action is discussed.     

Interactions of cyclic AMP and its dibutyryl analogue with model membrane: X-ray diffraction and Raman spectroscopic study using cubic liquid-crystalline phases of monoolein

Interactions of adenosine 3':5'-cyclic monophosphate (cAMP) and its dibutyryl analogue, N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (dbcAMP), with a lipid bilayer were studied by small-angle X-ray diffraction (SAXD) and Raman spectroscopy. The cubic Pn3m phase of monoolein (MO) served as a bilayer-based model system. SAXD measurements have indicated that incorporation of approximately 3 wt.% cAMP leaves the phase parameters practically unaltered, whereas the same content of dbcAMP induces the intercubic Pn3m-->Ia3d transition. By applying the concepts of lipid shape parameter and infinite periodic minimal surface to these MO phases, we have suggested that, as opposed to cAMP, dbcAMP associates with the MO bilayer. This conclusion has been supported by the different effects of phase matrix on the Raman shifts of the adenine and phosphate vibrational modes of these two nucleotides. Moreover, Raman spectra have indicated that dbcAMP inserts into the bilayer through the butyryladenine group, positioning dbcAMP preferentially at the polar/apolar interface.     

Three new potential cAMP affinity labels. Inactivation of human platelet low Km cAMP phosphodiesterase by 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate

Three new analogues of cAMP have been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (2-BDB-TcAMP), 2-[(3-bromo-2-oxopropyl)thio]-adenosine 3',5'-cyclic monophosphate (2-BOP-tcAMP), and 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (8-BDB-TcAMP). The bromoketo moiety has the ability to react with the nucleophilic side chains of several amino acids, while the dioxobutyl group can interact with arginine. These cAMP analogues were tested for their ability to inactivate the low Km (high affinity) cAMP phosphodiesterase from human platelets. The 2-BDB-TcAMP and 2-BOP-TcAMP were competitive inhibitors of cAMP hydrolysis by the phosphodiesterase with Ki values of 0.96 +/- 0.12 and 0.70 +/- 0.12 microM, respectively. However, 2-BDB-TcAMP and 2-BOP-TcAMP did not irreversibly inactivate the phosphodiesterase at pH values from 6.0 to 7.5 and at concentrations up to 10 mM. These results indicate that although the 2-substituted TcAMP analogues bind to the enzyme, there are no reactive amino acids in the vicinity of the 2-position of the cAMP binding site. In contrast, incubation of the platelet low Km cAMP phosphodiesterase with 8-BDB-TcAMP resulted in a time-dependent, irreversible inactivation of the enzyme with a second-order rate constant of 0.031 +/- 0.009 min-1 mM1. Addition of the substrates, cAMP and cGMP, and the product, AMP, to the reaction mixture resulted in marked decreases in the inactivation rate, suggesting that the inactivation was due to reaction at the active site of the phosphodiesterase.(ABSTRACT TRUNCATED AT 250 WORDS)