High Concentrations Of Cyclic AMP Research Articles

Exogenous dibutyryl cAMP affects meiotic maturation via protein kinase A activation; it stimulates further embryonic development including blastocyst quality in pigs

High concentrations of cyclic AMP in germinal vesicle oocytes generally inhibit GVBD. Thus, maintaining the GV stage in growing oocytes is essential for the developmental competence of the eggs. In this study, we traced the effects of dibutyryl cyclic AMP on meiotic maturation and early embryonic development in pigs. We also investigated several blastocyst qualities, including structural integrity, mitochondrial membrane potential, and apoptosis, which are affected by dbcAMP. To determine whether increased concentrations of cAMP inhibit GVBD, we explored the meiotic patterns and during maturation of pig oocytes. When treated with dbcAMP for 22h, 91.1% of the oocytes were arrested in the GV stage compared to only 38.8% of the oocytes in the control group (P<0.05). After completion of IVM, a higher proportion of the dbcAMP-treated oocytes were in metaphase II than the untreated ones (91.3% vs. 72.8%, P<0.05). Western blot analysis showed a reduction (at 22h) and/or increase (at 44h) in MPF and MAP kinase activities in porcine oocytes treated with dbcAMP for the first 22h of IVM compared to the untreated control. We also confirmed that protein kinase A activity increased in dbcAMP-treated oocytes, indicating an elevated intracellular concentration of cAMP. After IVF, the frequency of polyspermy in the dbcAMP-treated group decreased compared to that in the control group (22.4% vs. 47.4%, P<0.05). Furthermore, blastocyst formation, the blastocyst cell number, mitochondrial membrane potential, and apoptosis were enhanced and/or reduced by dbcAMP in both IVF and SCNT embryos. We concluded that synchronizing meiotic resumption by dbcAMP treatment improved the developmental capacity and embryonic qualities of IVF and SCNT embryos by increasing the mitochondrial membrane potential and decreasing the incidence of apoptosis in preimplantation-stage porcine embryos.

Functional roles of the two cyclic AMP-dependent forms of cyclic AMP receptor protein from Escherichia coli

The cyclic AMP receptor protein activates transcription in Escherichia coli, only when complexed with cyclic AMP. The cyclic AMP receptor protein-cyclic AMP complex formed at low concentrations of cyclic AMP has a different conformation from either cyclic AMP receptor protein alone or its complex with cyclic AMP formed at high cyclic AMP concentrations. Various biophysical data suggest that the latter complex resembles free cyclic AMP receptor protein. We have examined the conformational and biological properties of cyclic AMP receptor protein as a function of cyclic AMP concentrations, using the gal operon of E. coli. A biphasic behavior is observed. It is shown that only the complex formed at lower concentrations of cyclic AMP is the transcriptionally active form. This difference between the complexes at different levels of cyclic AMP arises from a decreased ability of the cyclic AMP receptor protein-cyclic AMP complex at high cyclic AMP concentrations to bind to DNA at specific sites.

Challenge with high concentrations of cyclic AMP induces transient changes in the cytosolic free calcium concentration in Dictyostelium discoideum.

Dictyostelium discoideum cells use cyclic AMP (cAMP) for chemotactic signaling as well as for differentiation. The precise regulation of the cytosolic Ca2+ concentration ([Ca2+]i) seems to play a key role for both processes. We performed single cell measurements of [Ca2+]i in amoebae that were starved in suspension for various times and scrape-loaded with the Ca2+ indicator fura-2. Stimulation of cells with cAMP at the concentration required to induce gene expression (> or = 100 microM) elicited a global transient increase in [Ca2+]i that depended on the presence of external Ca2+. Both vegetative and aggregation-competent cells displayed a rise in [Ca2+]i, with aggregation-competent cells responding more often than vegetative cells. Basal [Ca2+]i in the presence of Ca2+ was high in vegetative cells and declined during development; the cAMP-induced rise in [Ca2+]i was higher and lasted longer in vegetative cells than in aggregative cells. The addition of 2'-deoxy-cAMP, which binds to the cAMP receptor, induced an increase in [Ca2+]i, whereas the membrane-permeant analogue 8-bromo-cAMP that has a low affinity for the receptor but activates cAMP-dependent protein kinase had no effect. This indicates that the change in [Ca2+]i is mediated by the cell surface cAMP receptor. Since HC85 mutant cells, which lack the G alpha 2 subunit of the G-protein that couples the receptor to phospholipase C, also responded to stimulation with cAMP, the Ca2+ influx does not seem to be triggered by the phosphoinositide signaling cascade.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of plasma membrane-located calmodulin in the response decay of cyclic nucleotide-gated cation channel of cultured carrot cells

Increase in cytoplasmic cyclic AMP concentration stimulates Ca 2+ influx through the cyclic AMP-gated cation channel in the plasma membrane of cultured carrot cells. However, the Ca 2+ current terminated after a few minutes even in the presence of high concentrations of cyclic AMP indicating that hydrolysis of the nucleotide is not responsible for stop of the Ca 2+ influx. Cyclic AMP evoked discharge of Ca 2+ from inside-out sealed vesicles of carrot plasma membrane, and it was strongly inhibited when the suspension of the vesicles was supplemented with 1 μM of free Ca 2+, while Ca 2+ lower than 0.1 μM did not affect the Ca 2+-release. The Ca 2+ flux across plasma membrane was restored from this Ca 2+-induced inhibition by the addition of calmodulin inhibitors or anti-calmodulin. These results suggest that Ca 2+ influx initiated by the increase in intracellular cAMP in cultured carrot cells is terminated when the cytosolic Ca 2+ concentration reaches the excitatory level in the cells, and calmodulin located in the plasma membrane plays an important role in the response decay of the cyclic nucleotide-gated Ca 2+ channel.

Effects of cyclic AMP, glucocorticoids and insulin on the activities of phosphatidate phosphohydrolase, tyrosine aminotransferase and glycerol kinase in isolated rat hepatocytes in relation to the control of triacylglycerol synthesis and gluconeogenesis.

Rat hepatocytes were incubated in monolayer culture in modified Leibovitz L-15 medium containing either 10% (v/v) newborn-calf serum or 0.2% (w/v) fatty-acid-poor bovine serum albumin. The addition of 100 nM-dexamethasone increased the activities of both phosphatidate phosphohydrolase and tyrosine aminotransferase by about 3.5-fold after 8h, and these activities continued to rise until at least 24h. Incubating the hepatocytes in the albumin-containing medium with 10 microM- or 100 microM-8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate increased the activities of the phosphohydrolase and aminotransferase by 2.6- and 3.4-fold respectively after 8h. These increases were blocked by actinomycin D. The increases in the activities that were produced by the cyclic AMP analogue and dexamethasone were independent and approximately additive. Insulin when added alone did not alter the phosphohydrolase activity, but it increased the aminotransferase activity by 34%. The dexamethasone-induced increase in the phosphohydrolase activity was completely blocked by 7-144 microM-insulin, whereas that of the aminotransferase was only partly suppressed. Insulin had no significant Effects on the increases in the activities of phosphatidate phosphohydrolase and tyrosine aminotransferase that were produced by the cyclic AMP analogue, but this may be because the analogue is fairly resistant to degradation by the phosphodiesterase. The activity of glycerol kinase was not significantly changed by incubating the hepatocytes with insulin, dexamethasone and the cyclic AMP analogue alone or in combinations. It is proposed that high concentrations of cyclic AMP and glucocorticoids increase the total activity of phosphatidate phosphohydrolase in the liver and provide it with an increased capacity for synthesizing triacylglycerols and very-low-density lipoproteins, which is expressed when the availability of fatty acids is high. There appears to be a co-ordinated hormonal control of triacyglycerol synthesis and gluconeogenesis in diabetes and in metabolic stress to enable the liver to supply other organs with energy.

Effects of isoprenaline on the contraction-relaxation cycle in the cat trachea.

Effects of isoprenaline (Isop) on the contractile properties of the smooth muscle cells of cat trachea were investigated using intact and chemically skinned muscle preparations and an isometric tension recording method. In the intact muscle preparations, Isop 3 X 10(-10) or 3 X 10(-9) M significantly suppressed the amplitude of tonic contractions evoked by acetylcholine (ACh) 10(-7) M or 10(-5) M, respectively. Following treatment of the tissue with Ca2+-free 2 mM EGTA-containing solution after depletion of stored Ca2+ with caffeine, 2.5 mM Ca2+ was applied for 5 min (procedure 1), and subsequently 10 mM caffeine was applied in Ca2+-free 2 mM EGTA containing solution. The object was to estimate the amount of stored Ca2+ during procedure 1 from the amplitude of the caffeine (10 mM)-induced contraction (procedure 2). Isop, applied during procedure 1, did not affect the amplitude of the caffeine-induced contraction; however, when applied during procedure 2, this agent (10(-8)M) significantly suppressed the amplitude of the caffeine-induced contraction to about 90% of the control value. ACh (10(-5)M), applied during procedure 1, evoked phasic and tonic contractions. Isop (10(-8)M), applied simultaneously with ACh (10(-5)M), suppressed the amplitude of the ACh-induced contraction yet increased the amplitude of contraction evoked by the subsequent application of caffeine 10 mM (procedure 2). Effects of conditioning application of ACh (10(-7) or 10(-5)M) on the caffeine-induced contraction were observed in the presence or absence of Isop during procedure 2. When ACh 10(-5)M was used, subsequent application of caffeine 10 mM evoked no mechanical response, in control conditions. However, after the pretreatment of the tissue with Isop during procedure 2, the amplitude of the ACh (10(-5)M)-induced contraction was not affected, yet the subsequent application of caffeine (10 mM) evoked minute but discrete contractions, indicating that Isop did enhance the sequestration of free Ca2+ into the storage sites. In the saponin-treated skinned muscles, the minimum concentration of Ca2+ required to produce contraction was 1 X 10(-7)M, and the maximum contraction was obtained with 1 X 10(-5)M Ca2+. Isop (10(-6)M) had no effect on the relationship between free-Ca2+ and the amplitude of the contraction. However, simultaneous application of high concentrations of cyclic AMP (10(-4)M) and cyclic AMP-dependent protein kinase (50 micrograms ml-1) significantly suppressed contractions evoked by 3 X 10(-7) or 10(-5)M Ca2+. 8 These results indicate that Isop suppresses the contraction evoked by various agonists in the cat trachea, mainly through sequestration of Ca2+ into the intracellular storage sites, rather than by direct or indirect (through cyclic AMP) actions on the contractile proteins.

Phosphoproteins of the adrenal chromaffin granule membrane.

A fraction of chromaffin granule membranes contained a number of substrates for endogenous protein kinase activity as well as endogenous phosphatase activity. The major 32P-labelled polypeptide of molecular weight 43,000 appeared to be the alpha-subunit of pyruvate dehydrogenase of residual mitochondria. Several polypeptides showed cyclic AMP stimulation of phosphorylation of which the major polypeptide of molecular weight 59,000 shows half-maximal phosphorylation with 0.49 microM cyclic AMP. The phosphorylation of several other polypeptides is inhibited at high cyclic AMP concentrations. From studies with immunoprecipitation and two-dimensional electrophoresis it was found that alpha- and beta-tubulin and actin were absent from the granule membranes. However 32P labelling of a proportion of the copies of dopamine-beta-hydroxylase was demonstrated. The majority of the substrates for endogenous protein kinase activity are probably on the cytoplasmic side of the granule membrane.

Adenosine 3',5'-monophosphate waves in Dictyostelium discoideum: a demonstration by isotope dilution--fluorography.

The distribution of adenosine 3',5'-monophosphate (cyclic AMP) in fields of aggregating amoebae of Dictyostelium discoideum was examined by a novel isotope dilution-fluorographic technique. Cellular cyclic AMP was visualized by its competition with exogenous 3H-labeled cyclic AMP for high-affinity binding sites on protein kinase immobilized on a Millipore filter used to blot the monolayer. The cyclic AMP was distributed in spiral or concentric circular wave patterns which centered on the foci of the aggregations. These patterns were correlated with those of cell shape change that propagate through the monolayers: cells in regions of high concentrations of cyclic AMP were elongated (presumably moving up a cyclic AMP gradient), whereas those in regions of low cyclic AMP concentrations were randomly directed. The highest cyclic AMP concentrations were about 10(-6)M. The widths of the regions of elevated cyclic AMP were about 0.3 to 1 millimeter which, assuming a wave velocity of 300 micrometers per minute, suggests that a cell signals for about 1 to 3 minutes. These observations support the hypothesis that the aggregation process in Dictyostelium is mediated by the periodic relay of cyclic AMP signals and suggest a simple scheme for the dynamics of the aggregation process.

The effect of hemin, adenosine 3′,5′-monophosphate and phosphorylated sugars on Met-tRNA Metf deacylase activity in rabbit reticulocyte lysates

The effect of hemin, phosphorylated sugars, adenosine 3′,5′-monophosphate (cyclic AMP) and a number of purines on a specific initiator tRNA deacylase activity in rabbit reticulocytes has been investigated. In the concentration range established to be optimal for maximal stimulation of translation (5.5–30.0 μM), hemin produces a 20–82% inhibition of Met-tRNA Met f deacylation. In contrast, all phosphorylated sugars tested, with the exception of fructose 1,6-diphosphate, are without effect. High concentrations of cyclic AMP (2–4 mM) also significantly inhibit the deacylase activity. The role of hemin and Met-tRNA Met f deacylase in the control of peptide initiation are discussed.

Differential activation of type-I and type-II adenosine 3′:5′-cyclic monophosphate-dependent protein kinases in liver of glucagon-treated rats

The protein-bound cyclic AMP and the activity of cytosolic protein kinases in the presence and absence of cyclic AMP were determined in rat liver up to 2h after injection of glucagon. On the basis of the different salt-sensitivities of the activated cyclic AMP-dependent proteinkinases I and II, an activation of protein kinase II restricted to the high cyclic AMP concentrations present in the first 30 min after hormone injection was found. Essentially the same result was obtained by chromatographic analysis on DEAE-cellulose of liver cytosol from untreated rats and from rats killed at 2 and 60 min after glucagon injection. Protein kinase II activation was only detected at 2 min after injection. In contrast, the cyclic AMP-dependent protein kinase I was found to be nearly totally activated at 2 min and to be still almost as active at 60 min after the hormone stimulus, whereas the amount of bound cyclic AMP and the activation of total cytosolic protein kinases had fallen to two-thirds of their maximal values during this time period. A third cyclic AMP-independent protein kinase, which co-chromatographed with protein kinase type II, could be clearly distinguished from the two cyclic AMP-dependent kinases by use of the heat-stable inhibitor from bovine muscle, which totally inhibited the cyclic AMP-dependent enzymes, but stimulated the cyclic AMP-independent protein kinase.

Cyclic 3′:5′-nucleotide phosphodiesterase determined in various human tissues by deae-cellulose chromatography

Tissue extracts from human heart, lung, liver, kidney, skeletal muscle and cerebrum displayed at least 3 distinct cyclic 3′:5′-nucleotide phosphodieterase (EC 3.1.4.17) activity peaks (FI, FII, FIII) on DEAE-cellulose chromatography and various properties of these forms were compared in each tissue. FI eluted at about 0.08 M sodium acetate, hydrolyzed cyclic GMP more rapidly than it did cyclic AMP, and cyclic GMP hydrolysis by FI in most tissues was enhanced by a protein activator in the presence of CaCl 2. As only high concentrations of cyclic AMP inhibited cyclic GMP hydrolytic activity of FI, the enzyme probably has a low affinity for cyclic AMP. FII eluted at about 0.2 M sodium acetate, hydrolyzed both nucleotides at equal rates, and substrate affinities were relatively low. Cyclic GMP hydrolysis by FII was also stimulated by addition of a protein activator in the presence of CaCl 2 and cyclic AMP hydrolysis in this fraction was accelerated by a micromolar fraction of cyclic GMP. FIII eluted at about 0.35 M hydrolyzed cyclic AMP preferentially and was insensitive to protein activator. These two cyclic nucleotides act as mutual inhibitors of the hydrolysis in this fraction. Ratio of the cyclic GMP to cyclic AMP hydrolysis was in the order FI, FII, FIII. Four activity peaks were eluted from the cerebral extract and enzymes from this tissue exhibited much the same properties as observed in the other tissues examined herein.

Altered regulation of cycylic AMP-dependent protein kinase in a mouse lymphoma cell line

The ability of cyclic AMP to inhibit growth, cause cytolysis and induce synthesis of cyclic AMP-phosphodiesterase in S49.1 mouse lymphoma cells is deficient in cells selected on the basis of their resistance to killing by 2 mM dibutyryl cyclic AMP. The properties of the cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) in the cyclic AMP-sensitive (S) and cyclic AMP-resistant (R) lymphoma cells were comparatively studied. The cyclic AMP-dependent protein kinase activity of R cells cytosol exhibits an apparent K a for activation by cyclic AMP 100-fold greater than that of the enzyme from the parental S cells. The free regulatory and catalytic subunits from both S and R kinase are thermolabile, when associated in the holoenzyme the two subunits are more stable to heat inactivation in R kinase than in S kinase. The increased heat stability of R kinase is observed however only for the enzyme in which the catalytic and cyclic AMP-binding activities are expressed at high cyclic AMP concentrations (10 −5—10 −4 M), the activities expressed at low cyclic AMP concentrations (10 −9—10 −6 M) being thermolabile. The regulatory subunit of S kinase can be stabilized against heat inactivation by cyclic AMP binding both at 2 · 10 −7 and 10 −5 M cyclic AMP concentrations. In contrast, the regulatory subunit-cyclic AMP complex from R kinase is stable to heat inactivation only when formed in the presence of high cyclic AMP concentrations (10 −5 M). The findings indicate that the transition from a cyclic AMP-sensitive to a cyclic AMP-resistant lymphoma cell phenotype is related to a structural alteration in the regulatory subunit of the cyclic AMP-dependent protein kinase which has affected the protein's affinity for cyclic AMP and its interaction with the catalytic subunit.

Inhibitory action of noradrenaline and cyclic AMP in explants of rat cerebellum

IZNTOPHORETICALLY applied noradrenaline (NA) produces sustained depression of spontaneous firing of rat cerebellar Purkinje cells1–3 and this inhibition may be mediated by cyclic AMP4–5. This contention has been challenged by Godfraind and Pumain6 as well as by Lake and Jordan3 who failed to observe a depressant action of cyclic AMP. Because the results may be influenced by electrical current or pH artefacts, the kind of anaesthetics used or factors impeding the release of cyclic AMP from micropipettes7 we have used cultured Purkinje cells from rat cerebellum8–10 to test the hypothesis that cyclic AMP mediates the depressant action of NA on Purkinje cells. We found that high concentrations of cyclic AMP only slightly decreased the firing rate of Purkinje cells, but that low concentrations of inhibitors of phosphodiesterase strongly enhanced the inhibition induced by cyclic AMP and NA.

Adrenaline and cyclic AMP stimulated uptake of chloride and fluid secretion by isolated salivary glands of the lone star tick

1. Copper (10 −3 M) effectively inhibits 10 −5 M adrenaline stimulated uptake of 36Cl by isolated salivary glands of the lone star tick. 2. 2,4-dinitrophenol (DNP) (10 −3 M), ouabain (10 −3 M), and harmaline (10 −3 M) partially depress adrenaline stimulated uptake, but an antagonist of nerve postsynaptic β-receptors, propranolol (10 −3 M), is without effect. 3. Copper, DNP, and ouabain partially inhibit 10 −2 M cyclic AMP stimulated uptake of 36Cl by isolated tick salivary glands, but 10 −3 M harmaline enhances uptake. 4. A combination of high concentrations of cyclic AMP and theophylline stimulate fluid secretion by isolated tick salivary glands. 5. The significance of these findings to the control of ion movements into and across tick salivary glands and their significance mechanisms of fluid secretion is discussed.

Determination of limb bud chondrocytes during a transient block of the cell cycle

The process of determination is studied by using a cell culture system derived from dissociated chick embryo limb buds. When limb bud cells obtained from embryos younger than stage 25 (undertermined) are temporarily prevented from passing through the cell cycle (either by maintaining the cells on a petri dish or in the presence of high concentrations of cyclic AMP, both of which depress thymidine-H 3 incorporation), some cells subsequently form cartilage colonies. These results support the hypothesis that a temporary block at some stage in the cell cycle causes mesoblasts to acquire the capacity to differentiate into cartilage cells.

Amino acid transport and turnover of a transport system in liver slices from rats treated with glucagon and antibiotics

Experiments have been conducted in which glucagon-induced stimulation of α-aminoisobutyric acid (AIB) transport in rat liver, presumably mediated by cyclic AMP, was markedly inhibited by actinomycin D, cycloheximide or puromycin. Inhibition of transport occurred despite the presence of high concentrations of cyclic AMP, suggesting that the nucleotide may act prior to, or at the same point as, the antibiotics. Kinetic studies showed that 1) the half-life of the glucagon-stimulated system(s) was less than 1 hour, and 2) glucagon treatment doubled V max without having any effect on the apparent K m for hepatic AIB transport. The results suggest that glucagon stimulates hepatic AIB transport by increasing the synthesis of some critical protein having a rapid turnover rate; this effect may be due to a prior increase in RNA synthesis.

Studies on cyclic AMP phosphodiesterase in sea urchin eggs

1. 1.|Sea urchin eggs have multiple forms of cyclic AMP-phosphodiesterase (EC 3.1.4.17), 70% of which is localized in the post-microsomal supernatant. However, in fertilized eggs at about first cleavage, there is a significant decrease in the amount (55%) of the enzyme detected in the post-microsomal supernatant. This difference in the amount of phosphodiesterase present in the post-microsomal supernatant of cleaving eggs could be accounted for by a 2-fold increase in enzyme activity in heavy membrane fractions of cleaving eggs, as compared to that of unfertilized eggs. 2. 2.|Kinetic studies show two apparent K m values for low and high cyclic AMP concentrations, i.e. 2.5·10 −6 and 1.4·10 −3 M , with V max values of 3.7·10 −8 and 3.2·10 −6 M , respectively. 3. 3.|Mg 2+ is essential for the activity of the enzyme and the optimum pH is 7.60. Dibutyryl cyclic AMP, N 6- monobutyryl cyclic AMP and ADP were found to be inhibitory. Methylxanthines also inhibited the sea urchin enzyme. ATP was found to be the most potent physiological inhibitor of the sea urchin phosphodiesterase. 4. 4.|A 4–5 fold increase in specific activity of phosphodiesterase was obtained in a 30–60% (NH 4) 2SO 4 fraction of the whole homogenate. General properties of the partially purified enzyme were similar to those observed for the whole homogenate enzyme. 5. 5.|Only a very weak cyclic GMP-phosphodiesterase activity was found in these egg-homogenates, which could be due to a non-specific activity of cyclic AMP-phosphodiesterase.

Heterogeneity and Unusually High Affinity in the Interactions of Adenosine 3′:5′-Monophosphate with Specific Binding Proteins from Liver and Hepatoma Cells

Abstract The binding of adenosine 3':5'-monophosphate (cyclic AMP) to proteins has been further characterized in postmitochondrial supernatant fractions from rat liver and cultured hepatoma (HTC) cells. Possible explanations for the high binding affinity (Kd l 0.5 nm) and nonlinear Scatchard binding plots are considered. Examination of the membrane filtration method used to measure the binding indicates that essentially all of the protein in the extracts is retained and that nonspecific adsorption of cyclic AMP is not a serious problem under the conditions used. At high concentrations of cyclic AMP, the amount of binding increases linearly over a range of protein concentrations. The binding studied is highly selective for cyclic AMP as shown by competition studies with related compounds. The time required to reach binding equilibrium at 0° varies with the pH, the nature of the sample, and the cyclic AMP concentration. The observation of nonlinear Scatchard binding plots requires a wide range of cyclic AMP concentrations, and this in turn necessitates binding times of several hours or more to achieve equilibrium at all points. The kinetics of release of bound cyclic AMP are heterogeneous for both liver and HTC extracts, showing a range of half times from minutes to days. An evaluation of the instability of binding activity under assay conditions shows that this instability cannot cause the upward curvature seen in Scatchard plots, but it could account for the additional plateau or region of downward curvature observed with liver samples. Prior exposure of a liver sample to pH 4 or to elevated temperatures eliminates the component(s) responsible for the plateau and transforms the Scatchard binding curve at pH 4 into one showing only the upward curvature characteristic of HTC samples. The high affinity portion of the heterogeneous Scatchard plots is markedly reduced by increasing the salt concentration in the binding reaction at pH 4, but not at pH 6.5. Treatment with HgCl2 in the cold also produces some selective elimination of the higher affinity portion of the HTC Scatchard binding curve. Although these selective effects on different regions of the HTC Scatchard plot are consistent with the presence of multiple components with differing affinities for cyclic AMP, other explanations cannot be ruled out. Negatively cooperative interactions among identical binding sites would also account for the curvature, and these interactions might be sensitive to some of the agents used. In addition, theoretical considerations show that the interaction between a single binding component and the catalytic subunit of a protein kinase can generate nonlinear Scatchard plots similar to those observed.

Effects of decapitation, ether and pentobarbital on guanosine 3′,5′-phosphate and adenosine 3′,5′-phosphate levels in rat tissues

Cyclic GMP and cyclic AMP levels in eight different rat tissues were examined after animlas were immersed in liquid nitrogen. In order of decreasing concentration, cerebellu, kidney, lung and cerebral cortex contained the greatest quantities fo cyclic GMP. These tissues also contained relatively high concentrations of cyclic AMP. Compared to values in animals which were sacrificed in liquid nitrogen, levels of both nucleotides in many of the tissues examined were altered by decapitation or anesthesia with ether and pentobarbital. Decapitation increased the levels of both cyclic GMP and cyclic AMP in cerebellum, lung, heart, liver and skeletabl muscle. However, decapitation increased only cyclic AMP in cerebral cortex and kidney. Our previously reported high level of cyclic GMP in lung was attributed to ether anesthesia and surgical removal which increased the cyclic GMP content in lung, heart, testis and skeletal muscle. The effect of ether on cyclic GMP levels in lung and heart was blocked by pretreatment of animals with atropine which indicated that cholinergic agents increase cyclic GMP content in these tissues. Acetylcholine and carbachol in the presence of theophylline increased the accumulation of cyclic GMP in incubations of rat lung minces. Increases in cyclic GMP and cyclic AMP levels in cerebellum with ether anesthesia were prevented if rats were immersed in liquid nitrogen after anesthesis with ether. Anesthesia with pentobarbital decreased the levels of cyclic GMP in cerebellum and kidney and increased the nucleotide in heart, liver, testis and skeletal muscle compared to levels in tissues from animals immersed in liquid nitrogen. However, pentobarbital increased cyclic AMP levels in cerebellum and cerebral cortex and decreased the nucleotide in liver, kidney, testis and skeletal muscle. These studies provide a possible explanation for the variability in in vivo levels of cyclic GMP and cyclic AMP which have been previously reported. In addition, these studies support the hypothesis that the synthesis and degradation of cyclic AMP and cyclic GMP are regulated independently and not necessarily in a parallel or reciprocal manner. These studies also suggest that the increase accumulation of one cyclic nucleotide has no major effect on the synthesis and/or metabolism of the other; however, such interactions cannot be entirely excluded from the results of this study.

Cyclic AMP and cyclic GMP enhance growth of chick embryo fibroblasts.

SEVERAL recent reports suggest an important role for cyclic AMP (adenosine 3′,5′-cyclic monophosphate) in the regulation of fibroblast growth. Addition of high concentrations of cyclic AMP or its dibutyryl derivative to culture medium slows the rate of proliferation of several mammalian cell lines12. The intracellular concentration of cyclic AMP seems to be inversely proportional to the doubling time of the cell population3. In a culture approaching con-fluency, cell-to-cell contacts seem to induce an increase in intracellular concentration of cyclic AMP, which is assumed to mediate the observed decline in proliferation4,5. We will show that while certain cyclic purine nucleotides, including cyclic AMP, seem to be associated with control of proliferation of chick embryo fibroblasts, their effect, unlike that on 3T3 cells, is to enhance the proliferation of chick embryo fibroblasts. Cyclic GMP enhances cell proliferation optimally at a concentration as low as 1 µM