Changes in cyclic nucleotide levels during embryonic development of chick hearts

The effects of isoproterenol, acetylcholine (Ach), and adenosine, on cyclic AMP (cAMP) and cyclic GMP (cGMP) contents were examined in chick hearts at various stages of embryonic development. The basal cAMP content was highest (87.7 +/- 1.3 pmol/mg protein) in young (3-day) embryonic chick hearts and decreased during development (9.6 +/- 0.6 pmol/mg protein in 9-19-day-old hearts). On the other hand, the cGMP content was lowest (45.5 +/- 2.3 fmol/mg protein) in young (3-day) embryonic chick hearts and increased during development (338 +/- 15.0 fmol/mg protein in 14-19-day-old hearts). Iso increased the cAMP concentration in embryonic hearts at all ages. Ach and Ado had no effect on the cAMP content at all ages. However, the Isoproterenol-induced stimulation of cAMP was inhibited by Ach and Adenosine at all ages. In young embryonic hearts, Ach and Ado increased cGMP concentration only slightly, whereas these agents caused a substantial increase in cGMP concentration in the older hearts. Thus, there was a clear age difference in the effects of Ach and Adenosine on the cGMP and cAMP concentrations. Nitroprusside and hydrogen peroxide increased cGMP concentration in older hearts (greater than 5-day-old) but not in the 3-day-old embryonic hearts. Thus, guanylate cyclase activity may be low in young (3-day-old) hearts. It summary, the cGMP level is very low in young embryonic chick hearts, and increases markedly during development. The changes in cGMP are reciprocal to those of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

cAMP AND cAMP-STIMULATED PROTEIN PHOSPHORYLATION IN ZOOSPORES OF BROWN ALGAE1

Adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP) were detected at concentrations of 8–11 and 10–20 pmol · mg^?1 protein, respectively, in zoospores of a brown alga, Undaria pinnatifida (Harvey) Suringer. Cellular levels of these cyclic nucleotides did not substantially change during dark to light transition. cAMP-stimulated protein phosphorylation was found in soluble cell-free extracts of zoospores of Undaria pinnatifida and Laminaria angustata Kjellman.     

Adenosine-Elicited Accumulation of Adenosine 3'', 5''-Cyclic Monophosphate in the Chick Embryo Retina

The cyclic AMP level of 17-day-old chick embryo retina increased from 20 to 331 pmol/mg protein when the tissue was incubated for 20 min in the presence of 4-(3-butoxy-4-methoxybenzyl-2-imidozolinone) (RO 20-1724). The addition of 0.5 mM-3-isobutyl-1-methylxanthine (IBMX) or 0.5 units/ml of adenosine deaminase (EC 3.5.4.4) to the medium reduced the increase of cyclic AMP content from 20 to 100 pmol/mg protein. Dipyridamole did not interfere with the rise of the retinal cyclic AMP level observed with RO 20-1724. The EC50 of 6-amino-2-chloropurine riboside (2-chloroadenosine)-elicited accumulation of cyclic AMP of retinas incubated in the presence of RO 20-1724 plus adenosine deaminase was approximately 1 microM. When retina incubation was carried out in the presence of 0.5 mM-IBMX, the 2-chloroadenosine dose-response curve was shifted to the right two orders of magnitude. Maximal stimulation of the cyclic AMP level of 17-day-old chick embryo retina incubated in the presence of 0.5 mM-IBMX was observed at 1 mM-adenosine concentration. This effect was not blocked by dopamine antagonists. Guanosine and adenine did not affect the retinal cyclic AMP level. AMP and ATP had a slight stimulatory effect. Adenosine response of embryonic retina increased sharply from the 14th to the 17th embryonic day. A similar, but not identical adenosine effect was observed in cultured retina cells.     

On the presence of adenosine 3′, 5′ -cyclic monophosphate in moss (Funariahygrometrica)

Partially purified nucleotide fraction of moss containing [14C]-labelled putative adenosine 3′, 5′ -cyclic monophosphate (cAMP) and marker authentic [3H] -cAMP was characterized by chemical deamination and also by the enzymatic hydrolysis with beef heart cyclic nucleotide phosphodiesterase. A significant conversion of marker authentic [3H] -cAMP into [3H] -inosine 3′, 5′ -cyclic monophosphate (cIMP) and [3H] -5′ adenosine monophosphate was observed by respective treatments. In contrast, the [14C] -labelled putative cAMP from control and theophylline-treated moss tissue was insensitive to chemical deamination and enzymatic hydrolysis. Apparently, the [14C] -labelled product which comigrates with authentic [3H] -cAMP does not represent true cAMP. Both the methods employed for characterization of the labelled putative cAMP were sensitive enough to detect picomole quantities of authentic [3H] -cAMP. Lack of detectability of prelabelled [14C] -cAMP in our preparations implies that the tissue may contain authentic cyclic AMP below the picomole levels. Thus, the attributed physiological role to adenosine 3′, 5′ -cyclic monophosphate in moss tissue appears somewhat skeptical.     

Neurotrophic control of cyclic nucleotide levels during muscle differentiation in cell culture

The effects of chick brain–spinal cord extract on morphological development and cyclic nucleotide levels of cultured chick embryo skeletal muscle cells were determined. It had previously been shown that the extract stimulated morphological differentation, protein synthesis, and cholinesterase activity of muscle cells. Myoblasts fused earlier and an increase in number as well as diameter of myotubes were seen in the extract treated cultures. Cyclic nucleotides levels were higher (almost twice the controls for both adenosine 3′, 5′ -cyclic monophosphate and guanosine 3′, 5′ -cyclic monophosphate) and preceded their occurence in the control cultures. It was suggested that factor(s) in the extract interact with membrane receptor(s) to alter nucleotide levels which, in turn, allow the effects to be expressed.     

CHANGE IN THE ACTIVITY OF LIPASE IN SEA URCHIN EGGS AND EMBRYOS DURING EARLY DEVELOPMENT*

During the early development of the sea urchin, Anthocidaris crassispina, the activity of lipase was maintained at the same level as in unfertilized eggs until the mesenchymal blastula stage (20 hr culture at 20°C) and then increased gradually after gastrulation. The activity in the embryos kept in SO^2?₄-free artificial sea water changed in a similar manner to that in those kept in normal sea water, during the development until 36 hr of fertilization. At 48 hr, the activity in the embryos, which had developed to the permanent blastulae in SO^2?₄-free sea water, was markedly lower than in normal plutei and was similar to that in unfertilized eggs. The lipase activity in fertilized eggs 30 min after fertilization, which was almost the same as that in unfertilized eggs was found mainly to be localized in the precipitate fraction obtained by the centrifugation at 12,000 x g for 20 min, whereas the activity in unfertilized eggs was found in the precipitate by the centrifugation at 105,000 x g for 60 min. Ca²⁺, adenosine 3′, 5′-cyclic monophosphate (cAMP) and guanosine 3′, 5′-cyclic monophosphate (cGMP) had no effect on the lipase activity.     

Increase in levels of cyclic AMP during avian limb chondrogenesis in vitro.

In the present study the level of cAMP was measured during in vitro chondrogenesis of wing mesenchyme of stage 24 chick embryos and was found to increase significantly from 6.3 pmol/mg protein at the end of the first day of culture to 9.7 pmol/mg protein on the second day, when chondrogenic expression is first detected by the appearance of an Alcian blue staining extracellular matrix. Nonchondrogenic cultures derived from wings of stage 19 embryos had a lower level of cAMP (4.4 +/- 0.07 pmol/mg protein). The level of cAMP in intact wings was 4.5 +/- 0.4 pmol/mg protein and did not change between stages 19 through 25. The correlatin between increased levels of cAMP and the onset of chondrogenesis is consistent with a role of cAMP in the expression of differentiated functions in chondrocytes, as well as in some other cell types.     

Adenosine 3′,5′-cyclic monophosphate-binding protein in sea urchin eggs and embryos

Adenosine 3′,5′-cyclic monophosphate (cAMP) binds to high-molecular substances which are probably proteins, in homogenates of sea urchin eggs and embryos. The bound cAMP is exchangeable. Optimal pH for the binding capacity of the proteins with cAMP is 4.0, and is shifted to 5.0 in the presence of 5 mM caffeine. The level of bound cAMP increases steeply during 10 minutes of incubation. This is then followed by a less steep increase. The level of bound cAMP decreases in the presence of NaCl. The dissociation onstant between cAMP and the proteins in homogenates of unfertilized and ertilized eggs is about 10 nM, and the value in the embryos at the gastrula stage is lower than that in the unfertilized egg homogenate.     

Increase in Levels of Cyclic AMP During Avian Limb Chondrogenesis in vitro

In the present study the level of cAMP was measured during in vitro chondrogenesis of wing mesenchyme of stage 24 chick embryos and was found to increase significantly from 6.3 pmol/mg protein at the end of the first day of culture to 9.7 pmol/mg protein on the second day, when chondrogenic expression is first detected by the appearance of an Alcian blue staining extracellular matrix. Nonchondrogenic cultures derived from wings of stage 19 embryos had a lower level of cAMP (4.4 ± 0.07 pmol/mg protein). The level of CAMP in intact wings was 4.5 ± 0.4 pmol/mg protein and did not change between stages 19 trough 25. The correlation between increased levels of CAMP and the onset of chondrogenesis is consistent with a role of cAMP in the expression of differentiated functions in chondrocytes, as well as in some other cell types.     

Nitric oxide and downstream second messenger cGMP and cAMP enhance adipogenesis in primary human preadipocytes

Background aimsObesity is correlated with chronic low-grade inflammation. Thus the induction of inflammation could be used to stimulate adipose tissue formation in tissue-engineering approaches. As nitric oxide (NO) is a key regulator of inflammation, we investigated the effect of NO and its downstream signaling molecule guanosine 3′,5′-cyclic monophosphate (cGMP) as well as adenosine 3′,5′-cyclic monophosphate (cAMP) on preadipocytes in vitro.MethodsPreadipocytes were isolated from human subcutaneous adipose tissue, cultured until confluence, and differentiated. The NO donor diethylenetriamine (DETA)/NO (30–150 μm) was added during proliferation and differentiation. Additionally, cGMP/cAMP analogs 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP), 8-(4-chlorophenylthio)-guanosine 3′,5′-cyclic monophosphate (8-pCPT-cGMP) and 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP), and the adenylyl cyclase activator forskolin, specific guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and adenylyl cyclase inhibitor 2′-5′-dideoxyadenosine (ddA), were applied. Proliferation and differentiation were evaluated.ResultsDETA/NO in combination with the standard differentiation procedure significantly enhanced maturation of precursor cells to adipocytes. Proliferation, in contrast, was inhibited in the presence of NO. The application of cGMP and cAMP, respectively, increased pre-adipocyte differentiation to an even higher extent than NO. Inhibitors of the underlying pathways caused a significant decrease in adipogenic conversion.ConclusionsOur results support the application of NO donors during transplantation of preadipocytes in a 3-dimensional setting to accelerate and optimize differentiation. The results suggest that, instead of the rather instable and reactive molecule NO, the application of cGMP and cAMP would be even more effective because these substances have a stronger adipogenic effect on preadipocytes and a longer half-life than NO. Also, by applying inhibitors of the underlying pathways, the induced inflammatory condition could be regulated to the desired level.     

ORNITHINE AND GLUTAMIC ACID DECARBOXYLASE ACTIVITIES IN THE DEVELOPING CHICK RETINA

Abstract— γ-Aminobutyric acid was found in 6-day-old chick embryo retina; approx 20% was formed from putrescine and the remainder from glutamic acid. However, at the 18th embryonic day only 1% of the γ-aminobutyric acid synthesized in retina was derived from putrescine. These results show that γ-aminobutyric acid is synthesized in chick embryo retina prior to synaptogenesis, and that γ-aminobutyric acid is synthesized via two pathways. The first pathway involves the conversion of putrescine to γ-aminobutyric acid; the second path is dependent upon the conversion of glutamic acid to γ-aminobutyric acid, catalyzed by glutamic acid decarboxylase.     

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

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

Identification of cytidine 2′,3′-cyclic monophosphate and uridine 2′,3′-cyclic monophosphate in Pseudomonas fluorescens pfo-1 culture

Cytidine 2′,3′-cyclic monophosphate (2′,3′-cCMP) and uridine 2′,3′-cyclic monophosphate (2′,3′-cUMP) were isolated from Pseudomonas fluorescens pfo-1 cell extracts by semi-preparative reverse phase HPLC. The structures of the two compounds were confirmed by NMR and mass spectroscopy against commercially available authentic samples. Concentrations of both intracellular and extracellular 2′,3′-cCMP and 2′,3′-cUMP were determined. Addition of 2′,3′-cCMP and 2′,3′-cUMP to P. fluorescens pfo-1 culture did not significantly affect the level of biofilm formation in static liquid cultures.     

Quantitative phosphoproteomics reveals novel roles of cAMP in plants

3′,5′-cyclic adenosine monophosphate (cAMP) is finally recognized as an essential signaling molecule in plants where cAMP-dependent processes include responses to hormones and environmental stimuli. To better understand the role of 3′,5′-cAMP at the systems level, we have undertaken a phosphoproteomic analysis to elucidate the cAMP-dependent response of tobacco BY-2 cells. These cells overexpress a molecular “sponge” that buffers free intracellular cAMP level. The results show that, firstly, in vivo cAMP dampening profoundly affects the plant kinome and notably mitogen-activated protein kinases, receptor-like kinases, and calcium-dependent protein kinases, thereby modulating the cellular responses at the systems level. Secondly, buffering cAMP levels also affects mRNA processing through the modulation of the phosphorylation status of several RNA-binding proteins with roles in splicing, including many serine and arginine-rich proteins. Thirdly, cAMP-dependent phosphorylation targets appear to be conserved among plant species. Taken together, these findings are consistent with an ancient role of cAMP in mRNA processing and cellular programming and suggest that unperturbed cellular cAMP levels are essential for cellular homeostasis and signaling in plant cells.     

A conjugate of decyltriphenylphosphonium with plastoquinone can carry cyclic adenosine monophosphate,but not cyclic guanosine monophosphate,across artificial and natural membranes

The present study demonstrated for the first time the interaction between adenosine 3′,5′-cyclic monophosphate (cAMP), one of the most important signaling compounds in living organisms, and the mitochondria-targeted antioxidant plastoquinonyl-decyltriphenylphosphonium (SkQ1). The data obtained on model liquid membranes and human platelets revealed the ability of SkQ1 to selectively transport cAMP, but not guanosine 3′,5′-cyclic monophosphate (cGMP), across both artificial and natural membranes. In particular, SkQ1 elicited translocation of cAMP from the source to the receiving phase of a Pressman-type cell, while showing low activity with cGMP. Importantly, only conjugate with plastoquinone, but not dodecyl-triphenylphosphonium, was effective in carrying cAMP. In human platelets, SkQ1 also appeared to serve as a carrier of cAMP, but not cGMP, from outside to inside the cell, as measured by phosphorylation of the vasodilator stimulated phosphoprotein. The SkQ1-induced transfer of cAMP across the plasma membrane found here can be tentatively suggested to interfere with cAMP signaling pathways in living cells.     

THE DIRECT MEASUREMENT OF THE AXOPLASMIC TRANSPORT OF INDIVIDUAL RNA SPECIES: TRANSFER BUT NOT RIBOSOMAL RNA IS TRANSPORTED

Analysis of chick retinal and tectal RNA revealed that in addition to the major cytoplasmic RNAs (rRNA and tRNA), a number of the small mol wt nuclear RNAs (snRNAs) can also be detected. Subfractionation data indicated that one of these molecules, DD′, is of at least 95% nuclear location within the retina. Thus, very little, if any, of the retinal DD′ is available for axoplasmic transport from the retina into the optic nerve and tectum. Following intraocular injection of [³H]uridine, considerable incorporation of isotope into DD′ was observed within the optic tectum after 4, 8 and 16 days. This result indicates the presence of considerable local (i.e. tectal) synthesis. The specific activities of 29S, 18S and 5S rRNA and 4s tRNA relative to that of DD′ were measured in the optic tectum 8 and 16 days after the intraocular introduction of [³H]uridine. The same measurements were also made in intracranially injected animals. While the 29S/DD′, 18S/DD′ and 5S/DD′ specific activity ratios obtained were independent of the injection route, the 4S/DD′ ratio obtained from intraocularly injected animals was significantly greater (at least 2-fold) than that obtained from intracranially injected animals. Similar analysis was also performed with the optic nerve complex at 16 days post-injection with identical results. These results demonstrate that tRNA, but not rRNA, is transported from the retina into the optic nerve and tectum in the 2-day-old chicken.     

Cyclic nucleotide esterification: relationship to phosphate ring geometry and growth regulation in synchronized human lymphocytes.

Infrared spectra of neutral aqueous solutions of nucleoside 3′,5′-cyclic monophosphates indicate an increase in the antisymmetric phosphoryl stretching frequency to 1236 cm^?1 from 1215 cm^?1 in trimethylene cyclic phosphates. A further increase to 1242 cm^?1 accompanies esterification of the 2′-ribose hydroxyl. The O²′-esterified and 2′-deoxy cyclic nucleotides examined display both reduced kinase binding and altered phosphoryl stretching frequencies, suggesting that modification of the phosphate ring represents a common feature in decreased kinase activation. Reversible inhibition of mitosis in thymidine-synchronized human lymphocytes by 2 mmN⁶,O²′-dibutyryladenosine 3′,5′-cyclic monophosphate and N⁶-monobutyryladenosine 3′,5′-cyclic monophosphate was observed. However, adenosine 3′,5′-cyclic monophosphate, O²′-monobutyryladenosine 3′,5′-cyclic monophosphate, butyric acid, and ethyl butyrate had no effect on mitosis when present at 2 mm concentrations during S and G2. These results are consistent with hydrolysis of O²′-monobutyryladenosine 3′,5′-cyclic monophosphate and adenosine 3′,5′-cyclic monophosphate by esterase and phosphodiesterase enzymes and suggest that modification of the N⁶ amino group is necessary for the antimitotic activity of N⁶,O²′-dibutyryladenosine 3′, 5′-cyclic monophosphate.     

Regulation of membrane functions and fatty acid composition in Escherichia coli by cyclic AMP receptor protein.

Adenosine 3′,5′-cyclic monophosphate (cAMP) has been shown to be involved in regulating a number of membrane functions in Escherichia coli cells, including various respiratory and transport activities. In this report we show that this regulation is mediated by the cAMP receptor protein (CRP). In addition, data are presented which show that the cAMP-CRP system is involved in regulating the E. coli fatty acid composition.     

Role of phosphodiesterase 2 in growth and invasion of human malignant melanoma cells

Cyclic nucleotide phosphodiesterases (PDEs) regulate the intracellular concentrations and effects of adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP). The role of PDEs in malignant tumor cells is still uncertain. The role of PDEs, especially PDE2, in human malignant melanoma PMP cell line was examined in this study. In PMP cells, 8-bromo-cAMP, a cAMP analog, inhibited cell growth and invasion. However, 8-bromo-cGMP, a cGMP analog, had little or no effect. PDE2 and PDE4, but not PDE3, were expressed in PMP cells. Growth and invasion of PMP cells were inhibited by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), a specific PDE2 inhibitor, but not by rolipram, a specific PDE4 inhibitor. Moreover, cell growth and invasion were inhibited by transfection of small interfering RNAs (siRNAs) specific for PDE2A and a catalytically-dead mutant of PDE2A. After treating cells with EHNA or rolipram, intracellular cAMP concentrations were increased. Growth and invasion were stimulated by PKA14-22, a PKA inhibitor, and inhibited by N⁶-benzoyl-c AMP, a PKA specific cAMP analog, whereas 8-(4-chlorophenylthio)-2′-O-methyl-cAMP, an Epac specific cAMP analog, did not. Invasion, but not growth, was stimulated by A-kinase anchor protein (AKAP) St-Ht31 inhibitory peptide. Based on these results, PDE2 appears to play an important role in growth and invasion of the human malignant melanoma PMP cell line. Selectively suppressing PDE2 might possibly inhibit growth and invasion of other malignant tumor cell lines.     

Response of a dog kidney cell line (MDCK) to antidiuretic hormone (ADH) with special reference to activation of protein kinase

Antidiuretic hormone (ADH) increased the adenosine 3′,5′-cyclic monophosphate (cAMP) content of cultured cells of the MDCK line, derived from normal dog kidney, after activation of adenylate cyclase. Parathyroid hormone and insulin did not affect the cAMP content of the cells and ADH did not increase the cAMP content of HeLa or 3T6 cells. Thus the effect of ADH on MDCK cells was specific. ADH activated the protein kinase of MDCK cells. Conversion of cAMP-dependent protein kinase to a cAMP-independent catalytic subunit in the ADH-treated cells was demonstrated by glycerol density gradient centrifugation.