Regulation of cyclic GMP, cyclic AMP and lactate dehydrogenase by putative neurotransmitters in the C6 rat glioma cell line.

In C6 cells norepinephrine and dopamine caused transient increases in cyclic GMP and cyclic AMP, as well as an induction of lactate dehydrogenase. All of these responses were blocked by l-propranolol, suggesting mediation by a β-receptor. Phentolamine potentiated the NE-increased cAMP levels by 5-fold when NE was used at suboptimal doses, suggesting the presence of α-adrenergic receptors in C6 cells. Carbamylcholine decreased the levels of both cyclic nucleotides, with hexamethonium partially reversing the effect on cyclic GMP. Dibutyryl-cyclic GMP or carbamylcholine reduced catecholamine-induced cyclic AMP levels. Serotonin increased cyclic GMP levels 60% and decreased cyclic AMP levels 36%. Calcium- and magnesium-free media inhibited the norepinephrine-induced levels of cyclic GMP and cyclic AMP respectively.     

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

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

A 3′,5′ Cyclic AMP (cAMP) Phosphodiesterase Modulates cAMP Levels and Optimizes Competence in Haemophilus influenzae Rd

Changes in intracellular 3′,5′ cyclic AMP (cAMP) concentration regulate the development of natural competence in Haemophilus influenzae. In Escherichia coli, cAMP levels are modulated by a cAMP phosphodiesterase encoded by the cpdA gene. We have used several approaches to demonstrate that the homologous icc gene of H. influenzae encodes a functional cAMP phosphodiesterase and that this gene limits intracellular cAMP and thereby influences competence and other cAMP-dependent processes. In E. coli, expression of cloned icc reduced both cAMP-dependent sugar fermentation and β-galactosidase expression, as has been shown for cpdA. In H. influenzae, an icc null mutation increased cAMP-dependent sugar fermentation and competence development in strains where these processes are limited by mutations reducing cAMP synthesis. When endogenous production of cAMP was eliminated by a cya mutation, an icc strain was 10,000-fold more sensitive to exogenous cAMP than an icc⁺ strain. The icc strain showed moderately elevated competence under noninducing conditions, as expected, but had subnormal competence increases at onset of stationary phase in rich medium, and on transfer to a nutrient-limited medium, suggesting that excessive cAMP may interfere with induction. Consistent with this finding, a cya strain cultured in 1 mM cAMP failed to develop maximal competence on transfer to inducing conditions. Thus, by limiting cAMP levels, the H. influenzae cAMP phosphodiesterase may coordinate its responses to nutritional stress, ensuring optimal competence development.     

Binding of cyclic AMP and cyclic GMP to renal cortical homogenates. Relationship with phosphorylation

This study examined the binding of both cyclic AMP and cyclic GMP to receptor proteins in particulate and soluble subfractions of renal cortical homogenates from the golden hamster. The binding of both nucleotides was compared to subsequent effects of both nucleotides on the phosphorylation of histone from identical fractions. Cyclic AMP binding and cyclic AMP-dependent protein kinase activity predominated in the cytosol, with some binding and enzyme activity also detected in particulate fractions. Cyclic GMP and cyclic GMP-dependent protein kinase activity could only be demonstrated in cytosolic fractions and represented only 20-30% of cyclic AMP-dependent activity in this fraction. Binding of both nucleotides was highly specific, however, cyclic AMP showed some interaction with cyclic GMP binding. Evidence suggesting that each nucleotide interacts with a specific protein kinase was as follows: both the binding activity of the cyclic nucleotides and their combined protein kinase activity show additivity; cyclic AMP and cyclic GMP binding activity could be separated on sucrose gradients; cyclic AMP and cyclic GMP protein kinase activity could be separated with Sephadex G-100 chromatography, after preincubation of homogenate supernatants with either cyclic AMP or cyclic GMP. The results demonstrate the presence of both cyclic AMP- and cyclic GMP-dependent protein kinase in renal cortex.     

Initiation of multicellular differentiation in Dictyostelium discoideum is regulated by coronin A

Many biological systems respond to environmental changes by activating intracellular signaling cascades, resulting in an appropriate response. One such system is represented by the social amoeba Dictyostelium discoideum. When food sources become scarce, these unicellular cells can initiate a cAMP-driven multicellular aggregation program to ensure long-term survival. On starvation, the cells secrete conditioned medium factors that initiate cAMP signal transduction by inducing expression of genes such as cAMP receptors and adenylate cyclase. The mechanisms involved in the activation of the first pulses of cAMP release have been unclear. We here show a crucial role for the evolutionarily conserved protein coronin A in the initiation of the cAMP response. On starvation, coronin A–deficient cells failed to up-regulate the expression of cAMP-regulated genes, thereby failing to initiate development, despite a normal prestarvation response. Of importance, external addition of cAMP to coronin A–deficient cells resulted in normal chemotaxis and aggregate formation, thereby restoring the developmental program and suggesting a functional cAMP relay in the absence of coronin A. These results suggest that coronin A is dispensable for cAMP sensing, chemotaxis, and development per se but is part of a signal transduction cascade essential for system initiation leading to multicellular development in Dictyostelium.     

Regional alterations in cyclic nucleotide levels with acute and chronic morphine treatment.

Systemic morphine briefly elevated the caudate cyclic AMP level and subsequently depressed those levels in the substantia nigra and hypothalamus. Thalamic cAMP was unaffected within sixty minutes of the injection. Cyclic GMP was reduced in all four structures by thirty minutes. Tolerant animals evidenced increased cAMP levels in all but the hypothalamus and reduced cGMP in all four structures. A challenge injection of morphine elevated the two nucleotides briefly in the substantia nigra, depressed only cAMP in the hypothalamus and did not alter levels in the other structures.     

In vitro Tn7 mutagenesis of Haemophilus influenzae Rd and characterization of the role of atpA in transformation.

Haemophilus influenzae Rd is a gram-negative bacterium capable of natural DNA transformation. The competent state occurs naturally in late exponential growth or can be induced by a nutritional downshift or by transient anaerobiosis. The genes cya, crp, topA, and sxy (tfoX) are known to function in the regulation of competence development. The phosphoenolpyruvate:carbohydrate phosphotransferase system functions to maintain levels of cyclic AMP necessary for competence development but is not directly involved in regulation. The exact signal(s) for competence and the genes that mediate the signal(s) are still unknown. In an effort to find additional regulatory genes, H. influenzae Rd was mutated by using an in vitro Tn7 system and screened for mutants with a reduced ability to induce the competence-regulatory gene, comA. Insertions in atpA, a gene coding for the alpha subunit of the F1 cytoplasmic domain of the ATP synthase, reduce transformation frequencies about 20-fold and cause a significant reduction in expression of competence-regulatory genes, while the expression of constitutive competence genes is only minimally affected. In addition, we found that an insertion in atpB, which encodes the a subunit of the F0 membrane-spanning domain, has a similar effect on transformation frequencies.     

Studies on the specificity of immunohistochemical techniques for cyclic AMP and cyclic GMP.

Immunohistochemical studies employing antibodies against cyclic nucleotides indicate that cyclic AMP and cyclic GMP are localized to distinct subcellular sites. These antibodies, however, cross-react weakly with noncyclic nucleotides (eg. ATP, GTP), and therefore we investigated the speficity of the immunohistochemical technique. Slides of fetal nuclei exposed to gaseous nitrous acid demonstrated reduced immunofluorescence. The slides were then incubated with cyclic and noncyclic nucleotides, and restoration of distinct cyclic AMP and cyclic GMP staining pattern was achieved only with appropriate cyclic nucleotides. Antibodies that were used have a greater affinity for acetylated derivatives of cyclic nucleotides. By using a gas phase technique, tissue slices were acetylated and immunohistochemical staining intensity was compared with the effect of acetylation on antibody affinity for various nucleotides. Acetylation greatly increased affinity of cyclic AMP antibody for cyclic AMP but not other nucleotides, and greatly intensified cyclic AMP staining. Acetylation moderately increased affinity of cyclic GMP antibody for cyclic GMP, and moderately intensified cyclic GMP staining. Conclusion: Both nitrous acid and acetylation studies support the specificity of the immunohistochemical method for cyclic nucleotides.     

The effect of cyclic AMP on neuritic outgrowth in explant cultures of developing chick olfactory epithelium

The effect of cyclic AMP (cAMP) analogs and phosphodiesterase (PDE) inhibitors on neurite outgrowth was studied in explant cultures of olfactory neurons. Nasal pits from 5- or 6-day-old chick embryos were minced, explanted into culture dishes, and grown in a serum-free medium. One of the cyclic AMP analogs, dibutyryl cyclic AMP (dbcAMP) or 8-bromo-cyclic AMP (8-Br-cAMP), or one of the PDE inhibitors, theophylline or isobutylmethylxanthine (IBMX), was added to the culture medium. The explants were examined for neurite outgrowth after 2 days in vitro. Db-cAMP increased the number of explants expressing neurites by 25-35% over control cultures, whereas 8-Br-cAMP had essentially no effect at the same concentrations. Addition of dibutyryl cyclic GMP (dbcGMP) gave no increase in neurite outgrowth, thus indicating that the effect of enhancing neuritic growth is specific to cAMP and not cyclic nucleotides in general. The resulting increase in neurite outgrowth is due to the cyclic nucleotide component of dbcAMP, since both IBMX and theophylline, which elevate intracellular cAMP, also increased neurite outgrowth significantly. When forskolin was added to the culture medium, there was a trend to increased neurite outgrowth; this was significantly enhanced when a subthreshold concentration of theophylline was added in addition to the forskolin.     

Preparation of cyclic nucleotide antisera with thyroglobulin-cyclic nucleotide conjugates

Antisera to cyclic AMP and cyclic GMP were obtained by immunizing rabbits with antigens prepared by conjugating the 2'0-succinyl derivative of the cyclic nucleotides to thyroglobulin. The cyclic nucleotide-thyroglobulin conjugates were injected intradermally into multiple sites on the backs of the animals. This immunization procedure resulted in the production of antiserum in four of five animals capable of binding at a final serum dilution of greater than 1:10,000, 20% of the corresponding iodinated cyclic nucleotide derivative added. The antisera were also highly specific. The antiserum for cyclic AMP had a 2500-fold or greater relative affinity for cyclic AMP than other nucleotides or nucleosides, while that for cyclic GMP had a 5000-fold or greater affinity for 2'0 acetylated nucleotides or nucleosides except for acetylated cyclic IMP. The obstacles to measuring cyclic nucleotides, particularly cyclic GMP, in tissues were also overcome by refining and simplifying the methods for iodination, purification and assay. Furthermore, a "disequilibrium" incubation was developed as an alternative to the acetylation method to increase the sensitivity of the radioimmunoassay. Thus, the levels of both cyclic GMP and cyclic AMP can be determined rapidly and easily in the same tissue sample.     

Metabolism and excretion of exogenous adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate. Studies in the isolated perfused rat kidney and in the intact rat.

Isolated rat kidneys were perfused with a recirculating medium containing exogenous adenosine 3':5'-monophosphate (cyclic AMP) or guanosine 3':5'-monophosphate (cyclic GMP) at an initial concentration of 0.1 mM. Both cyclic nucleotides were rapidly removed from the perfusate. Urinary excretion accounted for about 20% and 40% of the respective cyclic AMP and cyclic GMP lost from the perfusate. The metabolism of the cyclic nucleotides was studied by 14C-labeled cyclic nucleotides in the perfusate. During 60 min, 30% of added cyclic [14C]AMP was metabolized to renal [14C]adenine nucleotides (ATP, ADP, and AMP) and 30% to perfusate [14C]uric acid. Similarly, 20% of cyclic[14C]GMP was metabolized to renal [14C]guanine nucleotides (GTP, GDP, and GMP) and 30% to perfusate [14C]uric acid. Urine contained principally unchanged 14C-labeled cyclic nucleotide. Addition of 0.1 mM cyclic AMP to the perfusate elevated the renal ATP and ADP contents 2-fold. Addition of 0.1 mM of either cyclic AMP or cyclic GMP to the perfusate also elevated the renal production of uric acid 2- to 3-fold. The production and distribution of metabolites of exogenous cyclic nucleotides were also studied in the intact rat. Within 60 min after injection, 3.3 mumol of either 14C-labeled cyclic AMP or cyclic GMP was cleared from the plasma. Kidney cortex and liver were the principal tissues for 14C accumulation. Urinary excretion accounted for about 20 and 45% of the cyclic [14C]AMP and cyclic [14C]GMP lost from the plasma, respectively. The 14C found in the kidney and liver was present almost entirely as the respective purine mono-, di-, and trinucleotides. The other principal metabolite was [14C]allantoin, found in the urine and, to a lesser extent, the liver. The urine contained mostly unchanged 14C-labeled cyclic nucleotide. Unlike the findings with the perfused kidney, [14C]uric acid was not a significant metabolite of the 14C-labeled cyclic nucleotides in these in vivo experiments.     

The content of cyclic nucleotides in an organ culture of normal and atherosclerosis-affected human aorta

Cyclic AMP and cyclic GMP content was measured in intima media of unaffected and atherosclerotic areas of human aorta in a short-term organ culture. It was demonstrated that during short-term cultivation the content of both cyclic nucleotides in tissues is constant. The cyclic AMP content in fatty streaks and atherosclerotic plaques is significantly (2 to 7-fold) lower than in unaffected intima. The cyclic GMP level in atherosclerotic lesions is 1.5 to 3-fold higher than in normal. The content of both cyclic nucleotides in the media underlying fatty streaks and atherosclerotic plaques is the same as in the normal tissue. The obtained data indicate serious disorders in the system of cyclic nucleotides during atherosclerosis.     

Quantitative Analysis of Purine Nucleotides Indicates That Purinosomes Increase de Novo Purine Biosynthesis

Enzymes in the de novo purine biosynthesis pathway are recruited to form a dynamic metabolic complex referred to as the purinosome. Previous studies have demonstrated that purinosome assembly responds to purine levels in culture medium. Purine-depleted medium or 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) treatment stimulates the purinosome assembly in HeLa cells. Here, several metabolomic technologies were applied to quantify the static cellular levels of purine nucleotides and measure the de novo biosynthesis rate of IMP, AMP, and GMP. Direct comparison of purinosome-rich cells (cultured in purine-depleted medium) and normal cells showed a 3-fold increase in IMP concentration in purinosome-rich cells and similar levels of AMP, GMP, and ratios of AMP/GMP and ATP/ADP for both. In addition, a higher level of IMP was also observed in HeLa cells treated with DMAT. Furthermore, increases in the de novo IMP/AMP/GMP biosynthetic flux rate under purine-depleted condition were observed. The synthetic enzymes, adenylosuccinate synthase (ADSS) and inosine monophosphate dehydrogenase (IMPDH), downstream of IMP were also shown to be part of the purinosome. Collectively, these results provide further evidence that purinosome assembly is directly related to activated de novo purine biosynthesis, consistent with the functionality of the purinosome.     

Amylase secretion by rabbit parotid gland. Role of cyclic AMP and cyclic GMP.

Amylase secretion and changes in the levels of cyclic AMP and GMP were studied in rabbit parotid gland slices incubated in vitro with a variety of neurohumoral transmitters, their analogs and inhibitors. Cyclic GMP levels increased 8-fold 5 min after exposure to carbachol (10(-4) M), without a change in cyclic AMP levels; amylase output also rose. These effects were completely inhibited by muscarinic blockade with atropine, but were unaffected by alpha-adrenergic blockade with phenoxybenzamine. Epinephrine (4 - 10(-5) M) produced a rapid increase in the levels of both cyclic nucleotides and in amylase release. The increase in cyclic GMP level was inhibited by previous exposure of the slices to phenoxybenzamine, while the cyclic AMP rise was prevented by the beta-blocking agent, propranolol. Pure alpha-adrenergic stimulation with methoxamine (4 - 10(-4) M) produced modest elevations in cyclic GMP content and amylase output, effects blocked by pre-treatment of slices with either atropine or phenoxybenzamine. At a concentration of 4 - 10(-6) M, isoproterenol (a beta-agonist) failed to affect cyclic GMP levels, but promptly stimulated increases in cyclic AMP levels, and after a short lag, amylase secretion. At a higher dose (4 - 10(-5) M) isoproterenol produced elevations in the levels of both nucleotides. The carbachol-induced effects on cyclic GMP content and amylase release were greatly potentiated by the addition of isoproterenol (4 - 10(-6) M). These data strongly suggest that cholinergic muscarinic agonists and alpha-adrenergic agonists stimulate amylase output in rabit parotid gland by mechanisms involving cyclic GMP. The atropine-sensitive intracellular events effected by alpha-stimulation may be dependent upon endogenous generation of acetylcholine. Both cyclic nucleotides seem to be required for the early rapid secretion of amylase. The unique responses achieved by the combination of carbachol and isoproterenol suggest that isoproterenol may increase the sensitivity of this tissue to the effects of cholinergic stimuli.     

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 CaCl2. 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 CaCl2 and cyclic AMP hydrolysis in this fraction was accelerated by a micromolar fraction of cyclic GMP. FII 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.     

Capturing cyclic nucleotides in action: snapshots from crystallographic studies

Fifty years ago, cyclic AMP was discovered as a second messenger of hormone action, heralding the age of signal transduction. Many cellular processes were found to be regulated by cAMP and the related cyclic GMP. Cyclic nucleotides function by binding to and activating their effectors - protein kinase A, protein kinase G, cyclic-nucleotide-regulated ion channels and the guanine nucleotide-exchange factor Epac. Recent structural insights have now made it possible to propose a general structural mechanism for how cyclic nucleotides regulate these proteins.