INFLUENCE OF DIVALENT CATIONS ON REGULATION OF CYCLIC GMP AND CYCLIC AMP LEVELS IN BRAIN TISSUE

—Depolarizing concentrations of K⁺ elevate levels of both adenosine 3′,5′monophosphate (cyclic AMP) and guanosine 3′,5′monophosphate (cyclic GMP) in incubated slices of mouse cerebellum. Calcium is an essential requirement for the K⁺ -induced accumulation of cyclic GMP. Barium and Sr²⁺, but not Mn²⁺ or Co²⁺, can substitute for Ca²⁺ in this process. Relatively high concentrations of Mg²⁺ inhibit the effect of Ca²⁺ on K⁺-induced accumulation of cyclic GMP. In contrast, depolarizing concentrations of K⁺ are capable of elevating cyclic AMP levels in brain slices suspended in media containing Mg²⁺ and no other divalent cations. High concentrations of Ca²⁺ (1 mm or greater) augment this Mg²⁺ -dependent, K⁺-induced accumulation of cyclic AMP, however. Strontium and Mn²⁺, but not Ba²⁺ or Co²⁺, can substitute for Ca²⁺ in this process, and high concentrations of Mg²⁺ are not inhibitory. The divalent cation ionophore, A-23187 (10 μm ), in the presence of extracellular Ca²⁺ elevates the level of cyclic GMP, but not cyclic AMP, in incubated mouse cerebellum slices. The results of this study indicate that intracellular Ca²⁺ concentration is a major factor regulating cyclic GMP levels in brain. In addition the present results suggest that, in brain tissue, depolarization-induced accumulation of cyclic GMP, but not cyclic AMP, is closely linked to some Ca²⁺-dependent mechanism(s) mediating release of intracellular substances.     

A study on sensing and adaptation in Dictyostelium discoideum: guanosine 3'',5''-phosphate accumulation and light-scattering responses

Cells of Dictyostelium discoideum respond to extracellular cyclic AMP with marked changes in intracellular cyclic GMP levels and light scattering. In this work, defined temporal increases in cyclic AMP were produced by the continuous addition of cyclic AMP to agitated suspensions of cells; concomitant hydrolysis of cyclic AMP by the cells subsequently established a constant, steady state concentration. The cells responded to the initial increase in extracellular cyclic AMP with a rapid increase in the intracellular cyclic GMP concentration and a rapid decrease in light scattering. At cyclic AMP input rates of 0.5-5 nM X s-1, the fast reactions of cyclic GMP and light scattering had already relaxed while the cyclic AMP concentration in the cell suspension was still increasing. The cells responded to constant concentrations of cyclic AMP with constant elevated cyclic GMP concentrations and constant decreased levels of light scattering. Our results are consistent with the existence of two types of perception systems, one of which adapts to constant stimuli and one of which does not adapt.     

Intracellular and Extracellular Cyclic Nucleotides in Wild-Type and White Collar Mutant Strains of Neurospora crassa: Temperature Dependent Efflux of Cyclic AMP from Mycelia

Cyclic AMP and cyclic GMP were released into the growth medium of mycelia of Neurospora crassa wild-type strains St.L.74A and Em5297a and by white collar-1 and white collar-2 mutant strains. After growth for 6 days at 18°C, there were 2.19 (St.L.74A), 5.83 (Em5297a), 1.38 (white collar-1), and 1.10 (white collar-2) nanomoles of cyclic AMP per gram dry weight of mycelia in the growth medium. These values corresponded to concentrations of cyclic AMP of between approximately 10 and 50 nanomolar. The corresponding values for extracellular cyclic GMP were typically less than 6% of the values for cyclic AMP. Following transfer to fresh medium, cyclic AMP efflux was demonstrated for each of the strains, and the amount of cyclic AMP exported into the fresh medium was greater at 25°C than 6°C. Intracellular cyclic AMP and cyclic GMP were also measured in each of the strains. The values for cyclic AMP were in the same range as those in the literature (approximately 0.5 to 1.5 nanomoles per gram dry weight of mycelia). However, the corresponding intracellular cyclic GMP values were less than 1% of the cyclic AMP values, i.e. more than 50 times lower than the value previously reported for the St.L.74A wild-type. Transfer of mycelia after 6 days at 18°C to fresh media and incubation for 2 hours at 25°C or 6°C did not consistently affect the intracellular level of cyclic AMP or cyclic GMP in the strains examined. We could detect no change in intracellular cyclic AMP when mycelia of the St.L.74A wild-type strain were irradiated with blue light for periods of up to 3.0 hours at 18°C, or in cyclic AMP and cyclic GMP for irradiation times of up to 1 minute at 6°C. We propose that the plasma membrane of Neurospora crassa is permeable to cyclic nucleotides, and the export of cyclic nucleotides into the growth medium may be a means of regulating intracellular levels. We conclude that three factors that affect carotenogenesis in Neurospora crassa (blue light, temperature, and the white collar mutations) have no appreciable effect on the total measurable intracellular cyclic nucleotides in this organism. There was no extracellular or intracellular cyclic AMP or cyclic GMP in the crisp-1 mutant strain, which suggested either that adenylate cyclase (which is absent in crisp-1) catalyzes the synthesis of both cyclic AMP and cyclic GMP or that the crisp-1 mutation somehow results in a deficiency of two enzymes (adenylate and guanylate cyclase).     

Evidence for a messenger function of cyclic GMP during phosphodiesterase induction in Dictyostelium discoideum

Chemotactic stimulation of vegetative or aggregative Dictyostelium discoideum cells induced a transient elevation of cyclic GMP levels. The addition of chemoattractants to postvegetative cells by pulsing induced phosphodiesterase activity. The following lines of evidence suggest a messenger function for cyclic GMP in the induction of phosphodiesterase: (i) Folic acid and cyclic AMP increased cyclic GMP levels and induced phosphodiesterase activity. (ii) Cyclic AMP induced both cyclic GMP accumulation and phosphodiesterase activity by binding to a rate receptor. (iii) The effects of chemical modification of cyclic AMP or folic acid on cyclic GMP accumulation and phosphodiesterase induction were closely correlated. (iv) A close correlation existed between the increase of cyclic GMP levels and the amount of phosphodiesterase induced, independent of the type of chemoattractant by which this cyclic GMP accumulation was produced. (v) Computer simulation of cyclic GMP binding to intracellular cyclic GMP-binding proteins indicates that half-maximal occupation by cyclic GMP required the same chemoattractant concentration as did half-maximal phosphodiesterase induction.     

Signal transduction for Schistocerca gregaria ion transport peptide is mediated via both cyclic AMP and cyclic GMP

The second messengers involved in the signal transduction for Schistocerca gregaria, ion transport peptide (Schgr-ITP) that regulates ion and fluid transport across the ileum of the desert locust S. gregaria, were measured using competitive enzyme-linked immunosorbent assays (ELISAs). Synthetic Schgr-ITP elevates intracellular levels of both cyclic AMP and cyclic GMP, measured over a 15 min period in the presence of 3-isobutyl-1-methylxanthine, in a dose-dependent manner. Furthermore, crude corpora cardiaca (CC) extracts elevate intracellular cyclic AMP levels 2-fold greater than Schgr-ITP, suggesting that factors present in the CC, other than Schgr-ITP, also act via this second messenger. These results suggest that the interaction of Schgr-ITP with two separate receptors, most likely a G-protein coupled receptor and a membrane bound guanylate cyclase, elevates intracellular levels of cyclic AMP and cyclic GMP to regulate ion and fluid transport across the locust ileum. Cyclic AMP stimulates Cl⁻, K⁺ and Na⁺ reabsorption, whereas secretion of H⁺ into the lumen of the ileum is most likely mediated via cyclic GMP. Cyclic GMP also stimulates Cl⁻ uptake in a similar manner to cyclic AMP. The measurement of tissue (central nervous system) levels of Schgr-ITP using an indirect ELISA confirms that the peptide is only present in the locust brain and the CC. The amounts present are greatest in the CC, where the peptide is presumably stored for release into the hemolymph when locusts feed.     

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

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

Species-Specific Aggregation Factor In Sponges

In dissociated single cells from the sponge Geodia cydonium, DNA synthesis is initiated after incubation with a homologous, soluble aggregation factor. During the DNA -initiation phase the cyclic AMP - and cyclic GMP levels vary drastically; the cyclic AMP content drops from 2.2 pmol/10⁶ cells to 0.3 pmol/10⁶ cells while the cyclic GMP content increases from 0.6 pmol to 3.7 pmol/10⁶ cells. the activity of neither the adenylate cyclase nor of the guanylate cyclase isolated from cells which have been incubated for different periods of time with the aggregation factor, is changed. the soluble as well as the particulate enzyme activities were checked in vitro. the cyclic nucleotide receptors have been isolated from the sponge cells and characterized with respect to their molecular weight, dissociation constant for cyclic AMP or cyclic GMP and intracellular concentration. None of these parameters are altered during aggregation factor-mediated DNA initiation. From these data it is concluded that the regulation of cyclic nucleotide levels is a consequence of a changed activity of nucleotide cyclases or of phosphodiesterases, but this is presumably not caused by a changed rate of synthesis of nucleotide cyclases or of cyclic nucleotide receptors.     

Effects of isoproterenol,theophylline and carbachol on cyclic nucleotide levels and relaxation of bovine tracheal smooth muscle

The effect of theophylline and isoproterenol on bovine tracheal smooth muscle tension and cyclic AMP levels was investigated. Concentrations of isoproterenol (4 × 10^?6 M) and theophylline (10 mM) that relaxed carbachol-contracted tracheal muscle by 85–95% did not significantly elevate control levels of cyclic AMP. In the absence of carbachol, several-fold increases in cyclic AMP were caused by isoproterenol although no elevations by theophylline were measurable. However, when isoproterenol and theophylline were administered together, theophylline potentiated the rise in cyclic AMP caused by isoproterenol. Phosphodiesterase studies in tracheal muscle showed the presence of a high and a low K_m enzyme which were inhibited by theophylline. Cyclic GMP levels were elevated in muscles contracted by carbachol as well as in carbachol-contracted muscles that had been relaxed by theophylline. In non-tension studies, in which the tracheal muscle was not under isometric tension, carbachol or theophylline alone increased cyclic GMP and together they synergistically elevated cyclic GMP. Atropine blocked the elevation caused by carbachol but not that caused by theophylline. In contrast to theophylline, isoproterenol did not elevate cyclic GMP, and in carbachol-contracted muscles that had been relaxed by isoproterenol, cyclic GMP levels were no different from control. Also, in non-tension studies, isoproterenol decreased basal cyclic GMP and antagonized the increase in cyclic GMP due to carbachol.The results indicate that whole-tissue levels of cyclic AMP and cyclic GMP do not correlate with the state of tracheal smooth muscle tension. Cyclic GMP levels do not clearly correlate with either contraction or relaxation. The inhibition by carbachol of increases in cyclic AMP due to isoproterenol and the inhibition by isoproterenol of increases in cyclic GMP due to carbachol provide evidence for a reciprocal cholinergic-adrenergic antagonism of cyclic AMP and cyclic GMP levels. The antagonism did not appear to be due to either cyclic nucleotide affecting the elevation of the other since the levels of both cyclic nucleotides were depressed.     

On the role of cyclic AMP and cyclic GMP in steroid production by bovine cortical cells

The time course of corticotropin-induced steroidogenesis and changes in intracellular cyclic AMP and cyclic GMP levels were investigated in isolated bovine adrenocortical cells prepared by trypsin digestion. Corticotropin produced a pea a peak rise in cyclic AMP during the first 5 min of stimulation and enhanced steroid production after 15 min. Corticotropin also caused a decrease in cortical cyclic GMP at 5 min; this decrease in cyclic GMP reverted to a 2–3 fold increase at 15–30 min which gradually subsided by 60 min. A steroidogenic concentration of prostaglandin E₂ also produced an elevation in the levels of both nucleotides, but the rise in cyclic GMP preceded the rise incyclic AMP. These results suggest that the relative amount of cyclic AMP and cyclic GMP, rather than the absolute levels of cyclic AMP, may be a key factor in the regulation of steroidogenesis.     

Two types of cyclic GMP binding site associated with the cyclic AMP-dependent protein kinase from lymphocytes

Low- and high-affinity binding sites for cyclic GMP were found to be associated with the cyclic AMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) from human tonsillar lymphocytes, but neither of them was identical with the cyclic AMP binding site.The enzyme activated by cyclic GMP phosphorylated the same site of calf thymus H2b histone as the cyclic AMP activated enzyme; however, more complex kinetics of activation were found with cyclic GMP.Two classes of cyclic GMP binding site were demonstrated by kinetic analysis of cyclic [³H]GMP binding in the enzyme preparations eluted by 0.1 M potassium phosphate (pH 7.0) from DEAE cellulose. The high-affinity cyclic GMP binding site (K_d about 44 · 10^?8 M belonged to some complex form of the protein kinase, as evidenced by the mutual inhibition of cyclic AMP binding and high affinity cyclic GMP binding. However, the high-affinity cyclic GMP binding site disappeared on Sephadex G-100 gel chromatography of the enzyme preparation, whereas the cyclic AMP binding activity was recovered quantitively as separate fractions. The low-affinity cyclic GMP binding site (K_d 2–5 · 10^?6 M) was demonstrated by the inhibitory effect of 10^?5 M cyclic GMP on cyclic AMP binding in each cyclic AMP binding fraction obtained by gel chromatography. However, cyclic AMP did not inhibit the binding of cyclic GMP to the low-affinity binding site.     

Partial purification of adenosine 3',5'-cyclic monophosphate phosphodiesterases from rat pancreas in the presence of excess protease inhibitors.

(i) Three forms of cyclic AMP phosphodiesterases (3′,5′-cyclic AMP 5′-nucleotidohydrolase, EC 3.1.4.17), F1, F2-I and F2-II, were partially purified from the soluble fraction of rat pancreas in the presence of excess protease inhibitors by DEAE-cellulose column chromatography and gel filtration and were characterized. (ii) F2-II, which was purified 31-fold, exhibited a single peak of activity on both polyacrylamide-gel electrophoresis and isoelectric focusing. The enzyme had a molecular weight of about 70,000, an isoelectric point of 3.9, and an optimal pH around 8.5 and required Mg²⁺ or Mn²⁺ but not Ca²⁺ for activity. The K_m values of this enzyme for cyclic AMP and cyclic GMP were 1 and 50 μm, respectively, while V values of this enzyme for cyclic AMP and cyclic GMP were 36.1 and 12.6 nmol min^?1 (mg of protein)^?1, respectively. Cyclic GMP competitively inhibited hydrolysis of cyclic AMP by this enzyme. Ro20-1724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone] also inhibited hydrolysis of cyclic AMP competitively, with a K_i value of 1 μm. (iii) Fraction F1, which was purified 10-fold, had a molecular weight of more than 500,000 and required Mg²⁺ for activity. Its K_m values for cyclic AMP were 1 and 5 μm. Its K_m value for cyclic GMP was 45 μm. Fraction F2-I, which was purified 26-fold, had a molecular weight of about 70,000. The ratio of the initial velocity of hydrolysis of cyclic GMP to that of cyclic AMP was 0.5 at a substrate concentration of 1 μm.     

The effect of isoproterenol and its analogs upon adenosine 3′,5′-monophosphate and guanosine 3′,5′-monophosphate levels in mouse parotid gland in vivo: Relationship to the stimulation of DNA synthesis

The ability of a large number of catecholamine analogs to stimulate DNA synthesis in the mouse parotid gland in vivo was compared to their effect on the levels of adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP) in this tissue. In the normal parotid gland the level of cyclic GMP is very low (10^?9 moles/kg wet wt), being only 1/800th of the cyclic AMP concentration. Isoproterenol increases the levels of cyclic AMP and cyclic GMP 30- and 3-fold, respectively. The increase in cyclic AMP is biphasic with an apparent early maximum at 2.5 min and a main peak at 15 min while the increase in cyclic GMP is monophasic with maximum levels at 15 min. Other analogs showed a similar effect on cyclic AMP levels but the time course of increases in cyclic GMP was very variable with peak stimulation as early as 1 min in some cases. The ability of analogs to cause the accumulation of cyclic AMP was correlated with their capacity to activate adenylate cyclase in parotid extracts and to act as β-adrenergic agonists in other systems. All compounds which raised cyclic AMP levels stimulated DNA synthesis but a number of other analogs also stimulated DNA synthesis. The effects of these analogs have been correlated with their ability to raise the intracellular concentration of cyclic GMP. Cholinergic agents also cause the accumulation of cyclic GMP but the effect of the analogs does not appear to be mediated through the cholinergic system as atropine does not block their effects and cholinergic agonists do not stimulate DNA synthesis. It is suggested that cholinergic agonists and the catecholamine analogs act primarily on the duct and acinar cells, respectively.Significant with inhibitors of the rises in cyclic nucleotide levels suggest that in isoproterenol stimulation it is the rise in cyclic GMP which is the more significant event in relation to stimulation of DNA synthesis.     

Hormone-induced changes in pyruvate kinase activity in isolated hepatocytes II. Relation to the hormonal regulation of gluconeogenesis gluconeogenesis

1. 1. Incubation of isolated hepatocytes with glucagon (10⁻⁶ M) or dibutyryl cyclic AMP (0.1 mM) causes a decrease in pyruvate kinase activity of 50%, measured at suboptimal substrate (phosphoenolpyruvate) concentrations and 1 mM Mg_free²⁺. The magnitude of the decrease in activity is not influenced by the applied extracellular concentrations of lactate (1 and 5 mM), glucose (5 and 30 mM) or fructose (10 and 25 mM). With all three substrates comparable inhibition percentages are induced by glucagon or dibutyryl cyclic AMP.

2. 2. The extent of inhibition of pyruvate kinase induced by incubation of hepatocytes with glucagon or dibutytyl cyclic AMP is not influenced by the extracellular Ca²⁺ concentration nor by the presence of 2 mM EGTA. The reactivation of pyruvate kinase seems to be inhibited by a high concentration of extracellular Ca²⁺ (2.6 mM) as compared to a low concentration of extracellular Ca²⁺ (0.26 mM).

3. 3. Incubation of hepatocytes in a Na⁺-free, high K⁺-concentration medium does not influence the magnitude of the pyruvate kinase inhibition induced by dibutyryl cyclic AMP. However, the reactivation reaction is stimulated under these incubation conditions.

4. 4. Incubation of hepatocytes with dibutyryl cyclic GMP (0.1 mM) leads to a 25% decrease in pyruvate kinase activity. The magnitude of the inhibition by dibutyryl cyclic (GMP) is not influenced by the presence of pyruvate (1 mM) or glucose (5 mM and 30 mM).

5. 5. The relative insensitivity of the pyruvate kinase inhibition induced by glucagon, dibutyryl cyclic AMP and dibutyryl cyclic GMP to the extracellular environment leads to the conclusion that the hormonal regulation of pyruvate kinase is not the only site of hormonal regulation of glycolysis and gluconeogenesis. It is concluded that hormonal regulation of pyruvate kinase activity is exerted by changes in the degree of (de)phosphorylation of the enzyme reflecting acute hormonal control as well as by changes in the concentration of the allosteric activator fructose 1,6-diphosphate. The latter depends at least in part on the hormonal control of the phosphofructokinase-fructose-1,6-phosphatase cycle.

The role of cyclic nucleotides and cell agglomeration in postaggregative enzyme synthesis in Dictyostelium discoideum.

Accumulation of cell-associated cyclic AMP phosphodiesterase and of two enzymes of carbohydrate metabolism, UDP glucose pyrophosphorylase and glycogen phosphorylase, was examined during development of Dictyostelium discoideum strain V12 M2 on agar and in suspension. In slow-shaken suspension, as well as on agar, phosphodiesterase began to accumulate about 3 hr after the initiation of development and reached a maximum approximately 2 hr later. At this time rapid accumulation of the two other enzymes, referred to as postaggregative enzymes, was initiated. In fast-shaken suspension phosphodiesterase accumulation did not stop, and synthesis of the postaggregative enzymes was greatly reduced. Large agglomerates formed shortly after the initiation of development in the slow-shaken culture, whereas in the fast-shaken culture the cells remained separate for about 5 hr and the agglomerates formed thereafter were much smaller. Addition of cyclic AMP (5 × 10^?4M) to the fast-shaken cells after 6 hr of development arrested further phosphodiesterase accumulation and greatly increased synthesis of the postaggregative enzymes. Cyclic GMP was considerably less effective. In contrast, cyclic AMP and cyclic GMP were equally effective in inducing accumulation of phosphodiesterase when added between 1 and 3 hr of development. We conclude that the synthesis of the two postaggregative enzymes studied here follows immediately upon the phase of phosphodiesterase synthesis and that both phases of synthesis depend in some way upon elevation of intracellular cyclic nucleotide levels.     

The uptake of cyclic AMP by human erythrocytes and its effect on membrane phosphorylation.

The effects of time and cyclic AMP concentration on cyclic AMP uptake and membrane phosphorylation were studied using intact human erythrocytes. The rate of uptake of cyclic [³H]AMP was nearly linear with respect to cyclic AMP concentration. The amount taken up was small compared to the extracellular cyclic AMP concentration, but was sufficient to significantly increase the intracellular cyclic AMP concentration. Incubation with cyclic AMP resulted in increased incorporation of ³²P_i into several phosphorylated membrane peptides of the intact erythrocytes. Although cyclic AMP altered the distribution of radioactivity among the membrane components, the total amount of incorporation was not increased. The effect of cyclic AMP on phosphorylation of membrane peptides was observed with extracellular cyclic AMP concentrations as low as 1 μm and was most pronounced in incubations of 1 to 4 h. These results indicate that cyclic AMP can enter erythrocytes in sufficient amounts to alter the activity of cyclic AMP-dependent protein kinases, or to alter the rate of turnover of certain phosphorylated membrane peptides.     

Calcium and cyclic nucleotide involvement in exocrine pancreatic enzyme secretion studied with the ionophore A-23187.

The ionophore, A-23187, was an effective pancreatic secretagogue. The response to A-23187 was Ca²⁺-dependent; Mg²⁺ reduced the secretory response to the ionophore. A-23187-stimulated enzyme release was potentiated by dibutyryl cyclic AMP; in the presence of carbachol, output of pancreatic protein paralleled the response to A-23187 alone. The time-course for secretion with A-23187 was similar to that observed with carbachol. The ionophore did not affect basal cyclic AMP levels but did stimulate a rapid Ca²⁺-dependent production of pancreatic cyclic GMP which preceded the onset of the secretory response. A-23187 did not significantly alter basal or carbachol-stimulated ⁴⁵Ca efflux from isotope preloaded glands; yet in Ca²⁺-lowered media, it inhibited (reversed) the secretory response to carbachol, an effect which may have been due to an outward transport by the ionophore of cholinergic-mobilized intracellular Ca²⁺. Like carbachol, A-23187, inhibits the incorporation of amino acid into new protein, the effect being partially dependent on extracellular Ca²⁺. The data suggest that the pancreatic cholinergic receptor acts as a Ca²⁺-ionophore and that extracellular Ca²⁺ is utilized in the synthesis of cyclic GMP.     

The adnosine 3',5'-monophosphate and guanosine 3',5'-monophosphate phosphodiesterases from human lung tissue.

Human lung tissue contains phosphodiesterase enzymes capable of hydrolyzing both adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP). The cyclic AMP enzyme exhibits three distinct binding affinities for its substrate (apparent Km = 0.4μM, 3μM, and 40μM) while the cyclic GMP enzyme reveals only two affinities (Km = 5μM and 40μM). The pH optima for the cyclic AMP and cyclic GMP phosphodiesterase are similar (pH 7.6–7.8). Both are inhibited by known inhibitors of phosphodiesterase activity (aminophylline, caffeine, and 3-isobutyl-1-methylxanthine). The divalent cations Mg²⁺ and Mn²⁺ stimulate cyclic AMP phosphodiesterase activity (in the absence of Mg²⁺) while Ca²⁺, Ni²⁺, and Cu²⁺ inhibit the enzyme. Histamine and imidazole slightly stimulate cyclic AMP hydrolytic activity. Thus, human lung tissue does contain multiple forms of both the cyclic AMP and cyclic GMP phosphodiesterase which are influenced by a variety of effectors.     

Variations in the levels of cyclic adenosine 3':5'-monophosphate and in the activities of adenylate cyclase and cyclic adenosine 3':5'-monophosphate phosphodiesterase during aerobic morphogenesis of Mucor rouxii

Particulate cell fractions of mycelium of Mucor rouxii contain adenylate cyclase activity which can be partially solubilized by 2% Lubrol PX. The enzyme requires Mn²⁺ and its activity is not modified by NaF or guanosine nucleotides. Mycelial extracts also contain cyclic adenosine 3′:5′-monophosphate phosphodiesterase activity, 60% of which is soluble. This activity shows characteristic low K_m (1 μm) for cyclic AMP and does not hydrolyze cyclic guanosine 3′:5′-monophosphate. It requires Mn²⁺ ions for maximal activity and is not inhibited by methylxanthines or activated by imidazole. Both enzymatic activities vary during the aerobic life cycle of the fungus. The spores have the highest levels of adenylate cyclase and cAMP phosphodiesterase, which decrease during the aerobic development. At the round cell stage, phosphodiesterase activity reaches 40% of the activity of the spores and varies only slightly thereafter. At this stage the specific activity of adenylate cyclase is 25% of the activity of ungerminated spores, and from this stage on, the activity increases up to the end of the logarithmic phase. Intracellular levels of cyclic AMP have been measured during aerobic germination. The variations of the intracellular level are tentatively explained by unequal variations in the activities of adenylate cyclase and cyclic AMP phosphodiesterase. A continuous increase of the extracellular cyclic AMP level during aerobic development has also been found, which cannot be accounted for solely by variations in the cyclase and diesterase activities.     

The accumulation of cyclic AMP and cyclic GMP in guinea pig brain slices: Effect of calcium ions,norepinephrine and adenosine

The effect of Ca²⁺ and putative neurotransmitters on formation of cyclic AMP and cyclic GMP has been studied in incubated slices of brain tissue. Cyclic AMP levels in cerebellar slices after about 90 min of incubation ranged from 10 pmol/mg protein in rabbit, to 25 in guinea pig, to 50 in mouse and 200 in rat. Cyclic GMP levels in the same four species showed no correlation with cyclic AMP levels and were, respectively, 1.3, 20, 5 and 30 pmol/mg protein. The absence of calcium during the prolonged incubation of cerebellar slices had little effect on final levels of cyclic AMP, while markedly decreasing final levels of cyclic GMP. Reintroduction of Ca²⁺ resulted in a rapid increase in cerebellar levels of cyclic GMP which was most pronounced for guinea pig where levels increased nearly 7-fold within 5 min. Prolonged incubation of guinea pig cerebral cortical slices in calcium-free medium greatly elevated cyclic AMP levels apparently through enhanced formation of adenosine, while having little effect on final levels of cyclic GMP. Norepinephrine and adenosine elicited accumulations of cyclic AMP and cyclic GMP in both guinea pig cerebral cortical and cerebellar slices. Glutamate, γ-aminobutyrate, glycine, carbachol, and phenylephrine at concentrations of 1 mM or less had little or noe effect on cyclic nucleotide levels in guinea pig cerebellar slices. Prostaglandin E₁ and histamine slightly increased cerebellar levels of cyclic AMP. Isoproterenol increased both cyclic AMP and cyclic GMP. The accumulation of cyclic AMP and cyclic GMP elicited by norepinephrine in cerebellar slices appeared, baed on dose vs. response curves, agonist-antaganonist relationships and calcium dependency, to involve in both cases activation of a similar set of ß-adrenergic receptors. In cerebellar slices accumulations of cyclic AMP and cyclic GMP elicted by norepinephrine and by a depolarizing agent, veratridine, were strongly dependent on the presence of calcium. The stimulatory effects of adenosine on cyclic AMP and cyclic GMP formation were antagonized by theophylline. The lack of correlations between levels of cyclic AMP and cyclic GMP under the various conditions suggested independent activation of cyclic AMP- and cyclic GMP-generating systems in guinea pig cerebellar slices by interactions with Ca²⁺, norephinephrine and adenosine.     

Cell differentiation in the absence of intracellular cyclic AMP pulses in Dictyostelium discoideum

Repeated additions of cyclic AMP to a morphogenetic mutant of Dictyostelium discoideum, agip 53, induced cell differentiation to the aggregation competent state as previously reported [Darmon, Brachet, and Pereira da Silva (1975). Proc. Nat. Acad. Sci. USA72, 3163–3166]. Cyclic AMP additions elicited transient increases of the intracellular cyclic GMP concentration but no significant increases of the intracellular cyclic AMP concentration. These results suggest that transient increases of the intracellular cyclic AMP concentration are not necessary for cell differentiation. Agip 53 seems to be unable to relay cyclic AMP signals. A defect in the receptor-mediated activation of adenylate cyclase could be the biochemical basis of the mutant phenotype of agip 53.