Demonstration, in leukemia L-1210 cells, of a phosphodiesterase acting on 3':5'-cyclic CMP but not on 3':5'-cyclic AMP or 3':5'-cyclic GMP.

cCMP-specific phosphodiesterase activity was demonstrated in the 80 to 100% ammonium sulfate fraction obtained from disrupted leukemia L-1210 cells. The activity was linear with time (up to 60 min), was a function of protein concentration, and was markedly stimulated by Mg2+ and by ammonium sulfate. Under identical assay conditions, no significant hydrolysis of cAMP or cGMP was observed, although these cyclic nucleotides served as substrates for phosphodiesterase(s) present in all the fractions obtained by less than 80% ammonium sulfate saturation. This is the first demonstration of a cCMP-specific phosphodiesterase.     

Neutrophil dysfunction in guanosine 3',5'-cyclic monophosphate-dependent protein kinase I-deficient mice

The regulation of neutrophil functions by Type I cGMP-dependent protein kinase (cGKI) was investigated in wild-type (WT) and cGKI-deficient (cGKI-/-) mice. We demonstrate that murine neutrophils expressed cGKIalpha. Similar to the regulation of Ca2+ by cGKI in other cells, there was a cGMP-dependent decrease in Ca2+ transients in response to C5a in WT, but not cGKI-/- bone marrow neutrophils. In vitro chemotaxis of bone marrow neutrophils to C5a or IL-8 was significantly greater in cGKI-/- than in WT. Enhanced chemotaxis was also observed with cGKI-/- peritoneal exudate neutrophils (PE-N). In vivo chemotaxis with an arachidonic acid-induced inflammatory ear model revealed an increase in both ear weight and myeloperoxidase (MPO) activity in ear punches of cGKI-/- vs WT mice. These changes were attributable to enhanced vascular permeability and increased neutrophil infiltration. The total extractable content of MPO, but not lysozyme, was significantly greater in cGKI-/- than in WT PE-N. Furthermore, the percentage of MPO released in response to fMLP from cGKI-/- (69%) was greater than that from WT PE-N (36%). PMA failed to induce MPO release from PE-N of either genotype. In contrast, fMLP and PMA released equivalent amounts of lysozyme from PE-N. However, the percentage released was less in cGKI-/- (approximately 60%) than in WT (approximately 90%) PE-N. Superoxide release (maximum velocity) revealed no genotype differences in responses to PMA or fMLP stimulation. In summary, these results show that cGKIalpha down-regulates Ca2+ transients and chemotaxis in murine neutrophils. The regulatory influences of cGKIalpha on the secretagogue responses are complex, depending on the granule subtype.     

Role of 3',5'-cyclic AMP in the control of nuclear protein kinase activity

The role of cAMP in the regulation of nuclear protein kinase activity was investigated. Acidic nuclear proteins prepared from rat liver nuclei were separated by phosphocellulose chromatography into four peaks of protein kinase activity and two peaks of cAMP-binding activity. A fraction which bound cAMP also inhibited the most active nuclear protein kinase, K IV, and the inhibition was diminished in the presence of 5 μM cAMP. Further support for the regulation of nuclear kinases by cAMP was obtained using a regulatory subunit prepared from rabbit muscle protein kinase. The muscle regulatory subunit markedly inhibited liver nuclear kinase activities. The addition of cAMP partially restored the activities.     

Characterization of a fluorescent substrate for the adenosine 3',5'-cyclic monophosphate-dependent protein kinase

A synthetic tetradecapeptide derived from the phosphorylation site of the beta-subunit of phosphorylase kinase (Arg-Thr-Lys-Arg-Ser-Gly-Ser-Val-Tyr-Glu-Pro-Leu-Lys-Ile) is a highly efficient substrate for the cAMP-dependent protein kinase, exhibiting a 36% decrease in the intrinsic tyrosine fluorescence on phosphorylation. The fluorescence changes in continuous assays were monitored to demonstrate the roles of protein kinase effectors (cAMP, the type II regulatory subunit, and the 8000-Da heat-stable inhibitor) in the regulation of the enzyme and to determine Km and Vmax. The phosphorylation reaction requires 1 mol ATP/mol peptide. Amino acid analysis demonstrates the presence of phosphoserine in the phosphorylated peptide. Auxiliary experiments show that tyrosine phosphorylation can also be detected fluorometrically and distinguished from serine or threonine phosphorylation.     

3',5'-cyclic adenosine monophosphate regulates expression of synaptotagmin in neonatal sympathetic ganglia in vitro

The expression of the synaptic vesicle protein, synaptotagmin, in developing rat superior cervical ganglia is influenced by transsynaptic factors associated with membrane depolarization. The present study examines the role of cyclic AMP in the regulation of synaptotagmin in neonatal superior cervical ganglia maintained in explant culture. Ganglia were treated for 48 h in vitro with the Na+-channel ionophore, veratridine, or with pharmacological agents that alter cyclic AMP levels. Levels of cyclic AMP and synaptotagmin were determined by radioimmunoassay. Veratridine treatment significantly increased cyclic AMP in cultured ganglia, with a long time course, and also increased synaptotagmin levels. Drugs that elevate cyclic AMP levels significantly increased synaptotagmin levels, with similar magnitude to that produced by veratridine treatment. These pharmacological agents did not alter neuron survival or total ganglionic protein content. No additive effects were observed after combined treatment with veratridine and pharmacological agents that increased cyclic AMP. Agents that blocked adenylyl cyclase blocked the veratridine-induced increase in synaptotagmin levels. The results suggest that regulation of expression of synaptotagmin in neonatal sympathetic neurons is mediated partially by cyclic AMP.     

3',5'-cyclic nucleotide phosphodiesterase from human brain

3':5'-Cyclic nucleotide phosphodiesterase was isolated from human brain and characterized. After the first stage of purification on phenyl-Sepharose, the enzyme activity was stimulated by Ca2+ and micromolar concentrations of cGMP. High pressure liquid chromatography on a DEAE-TSK-3SW column permitted to identify three ranges of enzymatic activity designated as PDE I, PDE II and PDE III. Neither of the three enzymes possessed a high selectivity for cAMP and cGMP substrates. The catalytic activity of PDE I and PDE II increased in the presence of Ca2+-calmodulin (up to 6-fold); the degradation of cAMP was decreased by cGMP. The Ca2+-calmodulin stimulated PDE I and PDE II activity was decreased by W-7. PDE I and PDE II can thus be classified as Ca2+-calmodulin-dependent phosphodiesterases. With cAMP as substrate, the PDE III activity increased in the presence of micromolar concentrations of cGMP (up to 10-fold), Ca2+ and endogenous calmodulin (up to 2-3-fold). No additivity in the effects of saturating concentrations of these compounds on PDE III was observed. Ca2+ did not influence the rate of cGMP hydrolysis catalyzed by PDE III. In comparison with PDE I and PDE II, the inhibition of PDE III was observed at higher concentrations of W-7 and was not limited by the basal level of the enzyme. These results do not provide any evidence in favour of the existence of several forms of the enzyme in the PDE III fraction. The double regulation of PDE III creates some difficulties for its classification.     

The effect of p,p'-dichlorodiphenyltrichloroethane on levels of guanosine 3',5'-cyclic monophosphate and adenosine 3',5'-cyclic monophosphate in two species of insects.

Within 1 h after topical application of a convulsive dose (4 mug per fly, 47 mg/kg) of p,p'-dichlorodiphenyltrichloroethane (DDT) to the adult male of Sarcophaga bullata Parker, guanosine 3',5'-cyclic monophosphate (cyclic GMP) levels rose by 71.5% (P less than 0.05) in the head, 159.5% (P less than 0.01) in the thorax, and 23.4% (P greater than 0.05) in the abdomen compared to controls. Adenosine 3',5'-cyclic monophosphate (cyclic AMP) levels were not significantly affected by the DDT treatment. A convulsive dose (100 mug per larva, 250 mg/kg) of DDT applied to larvae of Mamestra configurata Wlk. caused the whole body level of cyclic GMP to rise by 81.6% (P less than 0.01) after 1 h, and by 95.9% (P less than 0.01) after 3 h. Levels of cyclic AMP were not affected. A hypothesis is advanced suggesting that an abnormally high rate of discharge of acetylcholine (and in the later stages of poisoning, its actual accumulation) at central cholinergic synapses causes cyclic GMP levels to rise, perhaps in post-synaptic cells. The elevated cyclic GMP-cyclic AMP ratio found in DDT-poisoned insects may be of fundamental importance in the complex sequence of events leading to tremor, hyperexcitability, paralysis, and death.