Calmodulin differentially modulates the interleukin 1-induced biosynthesis of tissue inhibitor of metalloproteinases and matrix metalloproteinases in human uterine cervical fibroblasts.

The effects of a specific calmodulin inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on the synthesis of tissue inhibitor of metalloproteinases (TIMP) and precursor of matrix metalloproteinase 1/tissue collagenase (proMMP-1) and matrix metalloproteinase 3/stromelysin (proMMP-3), were examined using human uterine cervical fibroblasts in culture. When the cells were treated with human recombinant interleukin 1 alpha, the synthesis of TIMP, proMMP-1, and proMMP-3 was greatly enhanced along with the increase in the steady-state levels of mRNAs for respective proteins. The treatment of the cells with human recombinant interleukin 1 alpha and W-7 further augmented the production of proMMPs-1 and -3 and the accumulation of their mRNAs. In contrast, TIMP production and its steady-state mRNA level were reduced considerably under these conditions. Similar observations were made with another calmodulin inhibitor, trifluoperazine, but not with N-(6-aminohexyl)-1-naphthalenesulfonamide, the weakest inhibitor for calmodulin. These results indicate that calmodulin is required for the interleukin 1-enhanced synthesis of TIMP but it is a suppressor for the synthesis of proMMPs-1 and -3.     

Cyclic adenosine 3', 5'-monophosphate in Neisseria gonorrhoeae.

Intracellular cyclic adenosine 3', 5'-monophosphate (cAMP) was measured in two laboratory strains of Neisseria gonorrhoeae. Decreasing the glucose content of a defined media from 33 mM to 5.5 mM glucose resulted in an 11-to 25-fold increase of intracellular cAMP.     

Cyclic adenosine 3',5'-monophosphate and coryneform bacteria

Abstract We have detected cyclic AMP in the culture medium of amino acid-producing coryneform bacteria. Extracellular concentrations of cyclic AMP vary depending on the nutritional medium, the growth phase and the carbon source. It is also shown that the activity of Brevibacterium flavum adenylate cyclase in intact cells is stimulated in the presence of glucose. Furthermore, addition of cyclic AMP to B. flavum cultures decreased amino acid production similarly to the effect produced by the addition of inorganic phosphate.     

Cyclic adenosine 3':5'-monophosphate and the induction of deoxyribonucleic acid synthesis in liver.

A mixture containing glucagon and thyroid hormone was previously devised that enhances markedly nuclear DNA replication and mitosis in the parenchymal liver cells of the unoperated rat. It is now shown that the glucagon of the stimulatory solution can be completely replaced by a mixture of a butyryl derivative of cyclic adenosine 3':5'-monophosphate and theophylline. Cyclic guanosine 3':5'-monophosphate and its butyryl derivatives and insulin and high levels of glucose are inactive. The inactivity of N2-monobutyryl cyclic guanosine 3':5'-monophosphate cannot be ascribed to rapid breakdown in the animal or to the impenetrability of the liver cell since the coumpound elevates the rate of hepatic amino acid transport and the activity of ornithine decarboxylase. The observation of others (MacManus, J.P., Franks, D.J., Youdale, T. & Braceland, B.M. (1972) Biochem. Biophys. Res. Commun. 49, 1201-1207) that the level of cylcic adenosine 3':5'-monophosphate is raised during most of the prereplicative period after 70% hepatectomy is confirmed. The evidence supports a positive role for adenosine 3':5-monophosphate in regulating DNA synthesis in the liver.     

Cyclic adenosine 3':5'-monophosphate phosphodiesterase. Distinct forms in human lymphocytes and monocytes.

Adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterase activity of normal human peripheral blood leukocyte suspensions containing 90% lymphocytes and 10% monocytes showed anomalous kinetic behavior indicative of multiple enzyme forms. Kinetic analyses of purified lymphocyte (99%) or monocyte preparations (95%) indicated that only one type of phosphodiesterase was present in each cell type. None of the preparations contained any detectable guanosine 3':5'-monophosphate (cyclic GMP) hydrolytic activity. The lymphocyte enzyme had an apparent Km congruent to 0.4 muM for cyclic AMP and Vmax congruent to 0.5 picomoles/min/10(6) cells. These kinetic parameters were confirmed by several cell purification techniques used alone and sequentially. Sedimentation velocity analyses indicated that the higher Km monocyte enzyme had a molecular weight near 45,000 and that the lower Km lymphocyte enzyme most likely had a molecular weight near 98,000. A variety of procedures led to a loss of the higher molecular weight, high affinity enzyme leaving only the enzyme of 45,000 daltons with a much lower substrate affinity. A long term, stable human lymphoblastoid cell line had cyclic AMP phosphodiesterase activity that was similar to the lymphocyte enzyme by both physical and kinetic criteria. Lymphocyte cyclic AMP phosphodiesterase appears to be a soluble enzyme whose pH and temperature optima and cationic requirements are similar to those of other mammalian phosphodiesterases. The distinct cyclic AMP phosphodiesterase forms of these cells may possibly represent the basic, active subunit of mammalian cyclic nucleotide phosphodiesterases. We hypothesize that the extremely high affinity cyclic AMP phosphodiesterase of normal lymphocytes plays an important role in the regulation of normal function in these cells, and also in the rapid proliferative responses characteristic of the stimulated lymphocyte.     

Stimulation of cartilage macromolecule synthesis by adenosine 3',5'-monophosphate.

The role of cyclic AMP in the regulation of cartilage macromolecule synthesis in vitro was studied in pelvic cartilage from 10-12 day chick embryos. Incubation of cartilages in medium containing 0.5 mM cyclic AMP resulted in a 30% inhibition of 35SO4-2, [3H]leucine and [3H]uridine incorporation into proteoglycan, total protein and RNA, respectively. Higher concentrations of cyclic AMP had no greater effects. In contrast, butyrylated cyclic AMP derivatives (0.5-5.0 mM) added to the incubation medium stimulated (50-100%) the incorporation of these radiolabeled precursors into cartilage macromolecules. Theophylline, in concentrations (0.1-0.5 mM) which raise intracellular cyclic AMP, also increases the incorporation of radiolabeled precursors into macromolecules. The data indicate that exogenous cyclic AMP and butyrylated cyclic AMP derivatives have paradoxical effects on cartilage macromolecule synthesis. Butyrylated cyclic AMP derivatives, not exogenous cyclic AMP, mimic the effects of intracellular cyclic AMP. Incubation of embryonic chicken cartilage with exogenous cyclic AMP results in the extracellular degradation of the cyclic AMP to adenosine. Adenosine (0.125 mM) inhibits precursor incorporation into cartilage macromolecules. The metabolism of exogenous cyclic AMP generates sufficient adenosine to account for the observed inhibitory effects of exogenous cyclic AMP on cartilage macromolecule synthesis. Butyrylated cyclic AMP derivatives are not degraded during incubation with cartilage. The data indicate that cartilage is a tissue in which the effect of cyclic AMP is to stimulate anabolic processes.     

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.     

Compartmentalization of adenosine 3':5'-monophosphate and adenosine 3':5'-monophosphate-dependent protein kinase in heart tissue.

In rabbit heart homogenates about 50% of the cAMP-dependent protein kinase activity was associated with the low speed particulate fraction. In homogenates of rat or beef heart this fraction represented approximately 30% of the activity. The percentage of the enzyme in the particulate fraction was not appreciably affected either by preparing more dilute homogenates or by aging homogenates for up to 2 h before centrifugation. The particulate enzyme was not solubilized at physiological ionic strength or by the presence of exogenous proteins during homogenization. However, the holoenzyme or regulatory subunit could be solubilized either by Triton X-100, high pH, or trypsin treatment. In hearts of all species studied, the particulate-bound protein kinase was mainly or entirely the type II isozyme, suggesting isozyme compartmentalization. In rabbit hearts perfused in the absence of hormones and homogenized in the presence of 0.25 M NaCl, at least 50% of the cAMP in homogenates was associated with the particulate fraction. Omitting NaCl reduced the amount of particulate-bound cAMP. Most of the particulate-bound cAMP was probably associated with the regulatory subunit in this fraction since approximately 70% of the bound nucleotide was solubilized by addition of homogeneous catalytic subunit to the particulate fraction. The amount of cAMP in the particulate fraction (0.16 nmol/g of tissue) was approximately one-half the amount of the regulatory subunit monomer (0.31 nmol/g of tissue) in this fraction. The calculated amount of catalytic subunit in the particulate fraction was 0.18 nmol/g of tissue. Either epinephrine alone or epinephrine plus 1-methyl-3-isobutylxanthine increased the cAMP content of the particulate and supernatant fractions. The cAMP level was increased more in the supernatant fraction, possibly because the cAMP level became saturating for the regulatory subunit in the particulate fraction. The increase in cAMP was associated with translocation of a large percentage of the catalytic subunit activity from the particulate to the supernatant fraction. The distribution of the regulatory subunit of the enzyme was not significantly affected by this treatment. The catalytic subunit translocation could be mimicked by addition of cAMP to homogenates before centrifugation. The data suggest that the regulatory subunit of the protein kinase, at least that of isozyme II, is bound to particulate material, and theactive catalytic subunit is released by formation of the regulatory subunit-cAMP complex when the tissue cAMP concentration is elevated. A model for compartmentalized hormonal control is presented.     

Cyclic adenosine 3',5'-monophosphate and prostacyclin inhibit membrane phospholipase activity in platelets.

[¹⁴C]-Arachidonic acid is incorporated mainly into phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine of horse platelet membranes. Treatment of washed platelets with thrombin leads to a rapid loss of radioactivity from these phospholipids. The liberated [¹⁴C]-arachidonate is immediately transformed into hydroxyacids and thromboxanes. Treatment with dibutyryl cyclic AMP, cyclic AMP phosphodiesterase inhibitors or prostacyclin, a newly discovered prostaglandin that stimulates platelet adenylate cyclase, prevents the action of thrombin on phospholipid break-down as well as on platelet aggregation. Dibutyryl cyclic AMP does not affect the metabolism of exogenous [¹⁴C]-arachidonic acid. Cyclic AMP may thus play a crucial role in the regulation of platelet phospholipase acitivity, and this could explain at least in part the inhibition of aggregation caused by substances which, like prostacyclin, raise the levels of cyclic AMP.     

Cyclic adenosine 3',5'-monophosphate binding protein in developing myxospores of Myxococcus xanthus

The interaction of cyclic adenosine 3',5'-monophosphate (cAMP) with specific protein molecules was examined in the high-speed supernatant fraction of extracts made at stages throughout glycerol-induced myxospore development in Myxococcus xanthus. Experiments using 8-azido[32P]cAMP, a photoaffinity analogue of cAMP, and SDS - polyacrylamide gel electrophoresis showed that the nucleotide interacts with only a single protein band of 12 500 molecular weight. Both the identiy and amount of this protein remained constant throughout development. The binding protein was specific for cAMP; other nucleotides did not compete with cAMP for binding sites. A Scatchard analysis showed evidence of only a single class of binding sites with a high affinity for cAMP.