Phospholipid metabolism and lysosomal enzyme secretion by leukocytes. Effects of dibutyryl cyclic adenosine 3':5'-monophosphate and ATP.

The effects of dibutyryl cyclic adenosine 3':5'-monophosphate and ATP on isotope incorporation into phospholipids and the release of beta-glucuronidase into the extracellular medium were studied in polymorphonuclear leukocytes from guinea pig peritoneal exudates. Exogenous dibutyryl cyclic adenosine 3':5'-monophosphate (0.1--1.0 mM) reduced beta-glucoronidase release induced by cytochalasin B in the absence of inert particles. It selectively inhibited 32Pi incorporation into phosphatidic acid and the phosphoinositides and the incorporation of myo-[2-3H]inositol into the phosphoinositides. Added ATP (0.1--1.0 MM), but not other nucleotides, was found to potentiate beta-glucuronidase release provoked by cytochasin B, but it impaired the labeling of the phosphoinositides by myo-[2-3H]inositol. The mechanism of the inhibition the isotope incarparation into these acidic phospholipids by the two mucleotides has not been defined. Dibutyryl cyclic adenosine 3':5'-monophosphate at 2--4 mM concentration was not found to appreciably alter the incorporation of [gamma-32P]ATP into phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide.     

ATP increases chemoattractant induced cyclic GMP accumulation in Dictyostelium discoideum

Changes in guanosine cyclic 3′,5′-monophosphate associated with adenosine cyclic 3′,5′-monophosphate and folic acid addition in the presence of ATP have been examined in Dictyostelium discoideum. Preincubation with 1 mM ATP had no effect on the basal cyclic GMP level but increased the cycli GMP accumulation in response to cylci AMP (5·10⁻⁸ M) or folic acid (5·10⁻⁶ M) 40–50%. ATP could not be replaced by ADP of 5′-adenylyliminodiphosphate. Because ATP has no effect on cyclic AMP receptor binding these results indicate that structural membrane alterations (e.g. membrane phosphorylation) may control the transduction of a chemotactic signal.     

Formation of adenosine 3′,5′-monophosphate from adenosine in mouse thymocytes

The effect of adenosine on the mouse thymocyte adenylate cyclase-adenosine 3′:5′-monophosphate (cyclic AMP) system was examined. Adenosine, like prostaglandin E₁, can cause 5-fold or greater increases in thymocyte cyclic AMP content in the presence but not in the absence of certain cyclic phosphodiesterase inhibitors. Two non-methylxanthine inhibitors potentiated the prostaglandin E₁ and adenosine responses, while methylxanthines selectively inhibited the adenosine response. Adenosine increased cyclic AMP content significantly wihtin 1 min and was maximal by 10 to 20 min with approx. 2 and 10 μM adenosine being minimal and half-maximal effective doses, respectively. Combinations of prostaglandin E₁, isoproterenol and adenosine were near additive and not synergistic. Of the adenosine analogues tested, only 2-chloro- and 2-fluoroadenosine significantly increased cyclic AMP. Thymocytes prelabeled with [¹⁴C] adenine exhibited dramatic increases in cyclic [¹⁴C]AMP 10 min after addition of adenosine or prostaglandin E₁ which corresponded to simultaneously determined increases in total cyclic AMP. Using [¹⁴C]adenosine, the percent of total cyclic AMP increase due to adenosine was only 16%. Adenosine was also shown to elicit a 40% increase in particulate thymocyte adenylate cyclase activity. Therefore, the increased content of cyclic AMP seen in mouse thymocytes after incubation with adenosine was due primarily to stimulation of adenylate cyclase and only partially to conversion of adenosine to cyclic AMP. The increased cellular content of cyclic AMP may be, in part, responsible for various immunosuppressive effects of adenosine.     

The chemotactic effect of 5′-amido analogues of adenosine cyclic 3′,5′-monophosphate in the cellular slime moulds

Previously it was shown that amoebae of some Dictyostelium species are attracted by adenosine cyclic 3′,5′-monophosphate (cyclic AMP), and to a lesser extent, by the analogues of this nucleotide.We measured the chemotactic activity of several 5′-amido analogues of cyclic AMP by using a small population assay.Our investigations have shown unequivocally that the molecular receptor systems of cyclic AMP of the amoebae are highly sensitive to stereochemical alternation at the 5′position of the cyclophosphate ring, while the replacement of oxygen by nitrogen seems to exert no major influence. Alteration of the stereochemical envelope of the ring by a protruding group decisively alters the biological activity of the molecule, an effect which clearly does not depend on the type ot the group which protrudes.     

Effect of ascorbic acid on ACTH-induced cyclic AMP formation and steroidogenesis in isolated adrrenal cells of vitamin E-deficient rats

Isolated adrenal cells from Vitamin E-deficient and control rats were prepared by a trypsin digestion method. Cyclic adenosine 3′,5′-monophosphate (cyclic AMP) formation was studied in response to adrenocorticotropin (ACTH) in the presence and absence of ascorbate by measuring the conversion of prelabeled adenosine 5′-triphosphate [¹⁴C]ATP to cyclic [¹⁴C]AMP. Ascorbate (0.5 mM) inhibited ACTH-induced cyclic [¹⁴C]AMP formation in adrenal cells isolated from Vitamin E-deficient rats but had no effect in the control cells. The inhibitory effect of ascorbate on ACTH-induce cyclic AMP formation in Vitamin E-deficient rats decreased as the concentration of ACTH increased. In Vitamin E-deficient rats ascorbate inhibited ACTH-induced cyclic [¹⁴C]AMP formation after 30 min of incubation. There was no further significant accumulation of cyclic [¹⁴C]AMP at 60 min or 120 min although in the absence of ascorbate cyclic [¹⁴C]AMP continued to be formed. The in vitro addition of α-tocopherol reduced the inhibition of ACTH-induced cyclic [¹⁴C]AMP formation by ascorbate in Vitamin E-deficient rats.These studies suggest that α-tocopherol and ascorbate may affect ACTH-induced cyclic AMP formation through interaction with membrane-bound enzyme adenylate cyclase.     

Use of a vinyl phosphonate analog of ATP as a rotationally constrained probe of the C5′---O5′ torsion angle in ATP complexed to methionine adenosyl transferase

An analog of adenosine 5′-triphosphate (ATP) was synthesized in which the C4′---C5′---O---P^α system is replaced by a trans C4′---CH=CH---P^α system. In the form of 1:1 complexes with Mg, this analog and its counterpart with a C4′---CH₂---CH₂---P^α system were linear competitive inhibitors, with respect to MgATP, of the MAT-II (normal tissue) and MAT-T (hepatoma tissue) forms of rat ATP: -methionine-S-adenosyltransferase (MAT); K_m(ATP)/K_i values ranged from 0.4 to 2.4. 2′-Deoxy-ATP was a weak substrate, K_m(ATP)/K_m = 0.035, of MAT-II and a weak competitive inhibitor, K_m(ATP)/K_i = 0.07, of MAT-T. These findings, together with interactions of the MAT forms with other substrates and inhibitors, indicate that binding of ATP to these transferases is accompanied by little rotation about the C5′---O5′ bond, and that C4′ and P^α are in a trans-type relationship in enzyme-bound ATP.     

Stimulation of adenosine 3′,5′-monophosphate formation in mononuclear leukocytes by toad venoms

The content of adenosine 3′,5′-monophosphate in human mononuclear leukocytes was enhanced 3–5-times by venoms obtained from African toad (Bufo africanus), American toad (Bufo americanus), Colorado river toad (Bufo arenarum) and Marine toad (Bufo marinus) at 25 μg/ml for 5 min of incubation at 37°C. The maximum stimulation was observed after 1–5 min of incubation. The half-maximal stimulation was observed at 0.1 μg/ml venom obtained from Colorado river toad (Bufo arenarum). The increased content of adenosine 3′,5′-monophosphate in the mononuclear leukocytes persisted without significant change for at least 30 min of incubation at 37°C.     

2′-Azido-2′-deoxy- and 2′-amino-2′-deoxy-β-d-arabino-furanosyl purine nucleosides

A general method for the preparation of 2′-azido-2′-deoxy- and 2′-amino-2′-deoxyarabinofuranosyl-adenine and -guanine nucleosides is described. Selective benzoylation of 3-azido-3-deoxy-1,2-O-isopropylidene-α-d-glucofuranose afforded 3-azido-6-O-benzoyl-3-deoxy-1,2-O-isopropylidene-α-d-glucofuranose (1). Acid hydrolysis of 1, followed by oxidation with sodium metaperiodate and hydrolysis by sodium hydrogencarbonate gave 2-azido-2-deoxy-5-O-benzoyl-d-arabinofuranose (3), which was acetylated to give 1,3-di-O-acetyl-2-azido-5-O-benzoyl-2-deoxy-d-arabinofuranose (4). Compound 4 was converted into the 1-chlorides 5 and 6, which were condensed with silylated derivatives of 6-chloropurine and 2-acetamido-hypoxanthine. The condensation reaction gave α and β anomers of both 7- and 9-substituted purine nucleosides. The structures of the nucleosides were determined by n.m.r. and u.v. spectroscopy, and by correlation of the c.d. spectra of the newly prepared nucleosides with those published for known purine nucleosides.     

Regulation of DNA synthesis by guanosine-5′-diphosphate, cyclic guanosine-3′,5′-monophosphate, and cyclic adenosine-3′,5′-monophosphate in mouse lymphoid cells

The effect of various adenine and guanine nucleotides and nucleosides on DNA synthesis was studied in various types of mouse lymphoid cells. Two out of the ten compounds tested, namely guanosine-5′-diphosphate (GDP) and cyclic guanosine-3′,5′-monophosphate (cGMP) increased the thymidine incorporation into the DNA of the spleen cells and counteracted completely or partially the inhibitory action of cyclic adenosine-3′,5′-monophosphate (cAMP) on spleen cells stimulated by various B or T cell mitogens. GDP seems to act preferentially on thymus cells while cGMP acts better on bone marrow cells. The possible significance of the results for the mechanism of the mitogenic signal is discussed.     

Cyclic 3′,5′-AMP phosphodiesterase in Physarum polycephalum: II. Kinetic properties

The effect of several inhibitors of the enzyme cyclic 3′,5′-AMP phosphodiesterase as chemoattractants in Physarum polycephalum was examined. Of the compounds tested, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Roche 20-1724/001) and 1-ethyl-4-(isopropylidinehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid ethyl ester, hydrochloride (Squibb 20009) were the most potent attractants. 3-Isobutyl-1-methyl xanthine, theophylline, and morin (a flavanoid) were moderate attractants and sometimes gave negative chemotaxis at high concentrations. Cyclic 3′,5′-AMP was an effective, but not potent attractant. A repellent effect following the positive chemotactic action was sometimes observed with cyclic 3′,5′-AMP at concentrations as high as 1 · 10⁻² M. Dibutyryl cyclic AMP appeared to be a somewhat more potent attractant than cyclic 3′,5′-AMP. The 8-thiomethyl and 8-bromoderivatives of cyclic AMP, which are poorly hydrolyzed by the phosphodiesterase, were not attractants in Physarum. Possible participation of cyclic 3′,5′-AMP in the directional movement in P. polycephalum is discussed.     

Preparation and properties of 2′(or 3′)-O-(2,4,6-trinitrophenyl) adenosine 5′-triphosphate, an analog of adenosine triphosphate

An analog of adenosine triphosphate, 2′(or 3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate (TNP-ATP), was synthesized as a reporter-labeled substrate of heavy meromyosin ATPase. TNP-ATP was hydrolyzed by heavy meromyosin in the presence of CaCl₂ MgCl₂ or EDTA.TNP-ATP had absorption maxima at 259 nm, 408 nm and 470 nm at neutral pH. When bound to heavy meromyosin, TNP-ATP underwent the characteristic spectral shift. The difference spectrum resulting from the binding of TNP-ATP to heavy meromyosin at pH 8.0 had positive peaks at 415 nm and 518 nm, and a negative trough at 458 nm.The difference spectrum due to the binding of 2′(or 3′)-O-(2,4,6-trinitrophenyl)adenosine (TNP-adenosine) to heavy meromyosin had small positive peaks at 420 nm and 495 nm. This difference spectrum was similar to that of TNP-ATP or TNP-adenosine produced by 20% (v/v) ethyleneglycol perturbation. The positive peak at 495 nm in the difference spectrum due to the binding of TNP-adenosine to heavy meromyosin shifted toward 505 nm, when pyrophosphate or ATP was added to the reaction mixture.These results suggest that the difference spectrum of TNP-ATP due to the interaction with heavy meromyosin arises not only from the binding of the chromophoric portion of the TNP-ATP molecule but also from that of the phosphate portion.     

Studies on the presence of adenosine cyclic 3':5'-monophosphate in oat coleoptiles

The incorporation of adenosine-8-¹⁴C into adenosine cyclic 3′:5′-monophosphate in coleoptile-first leaf segments of Avena sativa L. was investigated. Homogenates of segments incubated in adenosine-8-¹⁴C for either 4 or 10 hours were partially purified by thin layer chromatography followed by paper electrophoresis. A radioactive fraction, less than 0.06% of the ¹⁴C present in the original homogenate, migrated as adenosine cyclic 3′:5′-monophosphate during electrophoresis. Upon treatment with cyclic nucleotide phosphodiesterase, however, less than 10% of this radioactive fraction appeared as 5′-AMP. Deamination with NaNO₂ as well as further chromatographical purification also suggested that only a small fraction of the ¹⁴C in the partially purified samples could be in adenosine cyclic 3′:5′-monophosphate. The data suggest that levels of this nucleotide can probably be no greater than 7 to 11 picomoles per gram of fresh weight in oat coleoptiles. Treatment of such coleoptiles with physiologically active concentrations of indoleacetic acid, furthermore, had no significant effect on the ¹⁴C radioactivity in marker adenosine cyclic 3′:5′-monophosphate-containing fractions at any stage of purification during several experiments.     

Furo[3′,2′:3,4]naphtho[1,2-d]imidazole derivatives as potential inhibitors of inflammatory factors in sepsis

Synthesis and anti-inflammatory effects of certain furo[3′,2′:3,4]naphtho[1,2-d]imidazole derivatives 12–18 were studied. These compounds were synthesized from naphtho[1,2-b]furan-4,5-dione (10) which in turn was prepared from the known 2-hydoxy-1,4-naphthoquinone (7) in a one pot reaction. Furo[3′,2′:3,4]naphtho[1,2-d]imidazole (12) was inactive (IC₅₀ value of >30 μM) while its 5-phenyl derivative 13, with an IC₅₀ value of 16.3 and 11.4 μM against lysozyme and β-glucuronidase release, respectively, was comparable to the positive trifluoperazine. The same potency was observed for 5-furan derivative 16 with an IC₅₀ value of 19.5 and 11.3 μM against lysozyme and β-glucuronidase release, respectively. An electron-withdrawing NO₂ substituted on 5-phenyl or 5-furanyl group led to the devoid of activity as in the cases of 14 and 17. Among them, compound 15 exhibited significant inhibitory effects, with an IC₅₀ value of 7.4 and 5.0 μM against lysozyme and β-glucuronidase release, respectively.For the LPS-induced NO production, the phenyl derivatives 12–15 were inactive while the nitrofuran counterparts 17 and 18 suppress LPS-induced NO production significantly, with an IC₅₀ value of 1.5 and 1.3 μM, respectively, which are more active than that of the positive 1400 W. Compounds 16–18 were capable of inhibiting LPS-induced iNOS protein expression at a dose-dependent manner in which compound 18, with an IC₅₀ of 0.52 μM in the inhibition of iNOS expression, is approximately fivefold more potent than that of the positive 1400 W. In the CLP rat animal model, compound 18 was found to be more active than the positive hydrocortisone in the inhibition of the iNOS mRNA expression in rat lung tissue. The sepsis-induced PGE2 production in rat serum decreased 150% by the pretreatment of 18 in a dose of 10 mg/kg.     

Biological activities and spectroscopic properties of chromophoric and fluorescent analogs of adenine nucleoside and nucleotides, 2′,3′-O-(2,4,6-trinitrocyclohexadienylidene) adenosine derivatives

The ribose-modified chromophoric and fluorescent analog of ATP 2′,3′-O-(2,4,6-trinitrocyclohexadienylidene) adenosine 5′-triphosphate (TNP-ATP) has been synthesized previously (Hiratsuka, T., and Uchida, K. (1973) Biochim. Biophys. Acta 320, 635–647 and Hiratsuka, T. (1976) Biochim. Biophys. Acta 453, 293–297). In the present study, four TNP-derivatives of ATP, ADP, AMP and adenosine were synthesized and compared for several chemical, spectral and enzymatic properties. Their visible absorption and fluorescent properties were found to be quite similar. Visible absorption and fluorescence spectra of TNP-derivatives were sensitive to solvent polarity. TNP-adenosine and TNP-AMP showed considerable substrate activities with adenosine deaminase and alkaline phosphatase, respectively. TNP-ATP proved to be an excellent substitute for ATP in adenylate kinase and myosin ATPase systems. The results indicate that these analogs are useful as chromophoric and fluorescent probes for hydrophobic regions in adenine nucleoside and nucleotide requiring enzymes.     

Endogenous synthesis of prostaglandins E2 and I2 in 3T3 fibroblasts transformed by polyoma virus: Effects on adenosine 3′:5′-monophosphate production

Prostaglandins (PG)E₁, E₂ and I₂ were produced by polyoma virus transformed (py) 3T3 fibroblasts. The levels of PGE₁, PGE₂ and 6-keto-PGF_1α (degradation product of PGI₂) were 22.7, 225 and 33.2 ng/ml medium, respectively, 72 h after medium change. The stimulatory potencies of exogenous PGE₁, PGE₂ and PGI₂ on adenosine 3′:5′-monophosphate (cyclic AMP) formation were similar. Therefore, the prostaglandin mediated increase in cyclic AMP levels observed during growth of these cells (Claesson, H.-E., Lindgren, J.Å. and Hammarström, S. (1977) Eur. J. Biochem. , 13) is largely (>80%) mediated by PGE₂ and to lesser extents by PGE₁ and PGI₂.     

Epidermal growth factor stimulates the incorporation of phosphate into phosphatidic acid and phosphoinositides but does not affect phosphoinositide breakdown by phospholipase C in renal cortical slices

The effects of epidermal growth factor (EGF) on the metabolism of phosphatidic acid and phosphoinositides were examined using renal cortical slices labelled with either sodium [³²P]orthophosphate or myo-[³H]inositol. EGF was found to increase the incorporation of phosphate into phosphatidic acid and phosphoinositides. This effect is not dependent on external calcium and is inhibited by 12-O-tetradecanoylphorbol 13-acetate (TPA). When phospholipids were prelabelled, EGF did not decrease the level of ³²P in phosphatidic acid and phosphoinositides, and EGF did not affect the formation of inositol phosphates or the concentration of cAMP and cGMP in renal tissue. The results show that EGF stimulates the incorporation of phosphate into phosphatidic acid and phosphoinositides, but does not affect breakdown of phosphoinositides by phospholipase C in renal cortical slices.     

The effects of fatty acids on phosphoinositide synthesis and myo-inositol accumulation in exocrine pancreas

The effects of arachidonic acid (20:4) on phosphoinositide turnover were examined in rat pancreatic acinar cells prelabeled with myo-[3H]inositol. Arachidonic acid (50 microM) increased the accumulation of myo-[3H]inositol, but not that of [3H]inositol monophosphate, [3H]inositol bisphosphate, or [3H]inositol trisphosphate. By contrast, 10 microM carbamoylcholine increased the accumulation of all four compounds. A combination of arachidonic acid plus carbamoylcholine caused a selective and marked accumulation of myo-[3H]inositol, which was abolished by 10 mM LiCl. Arachidonic acid (10-100 microM) produced a concentration-dependent inhibition of myo-[3H]inositol incorporation into phosphoinositides and markedly depressed carbamoylcholine-induced increases in myo-[3H]inositol incorporation into inositol phospholipids. Several other unsaturated and saturated fatty acids failed to elicit a synergistic response with carbamoylcholine in stimulating myo-[3H]inositol accumulation and did not retard the incorporation of myo-[3H]inositol into phosphoinositides. The fact that eicosapentaenoic acid (20:5), but not arachidic acid (20:0), mimicked the depressant effect of arachidonate on phosphoinositide labeling suggests that the degree of unsaturation of the fatty acid, rather than chain length, is important for inhibition of phosphoinositide synthesis. The arachidonate-induced decrease in myo-[3H]inositol incorporation was accompanied by a reduction in the steady state level of [32P]phosphatidylinositol 4,5-bisphosphate. The mass of arachidonic acid liberated in response to carbamoylcholine was measured by gas chromatography-mass spectrometry, and the time course of stimulated arachidonate accumulation paralleled that of inositol phosphate accumulation and amylase release. These observations suggest that in exocrine pancreas, endogenous arachidonic acid serves as a negative feedback regulator of phosphoinositide turnover.     

An easy and rapid method for the measurement of [γ-P]ATP specific radioactivity in tissue extracts obtained from in vitro rat heart preparations labeled with Pi

A rapid method for the measurement of [γ-³²P]ATP specific radioactivity in tissue extracts containing other ³²P-labeled compounds is described. The neutralized acid extract is incubated with cyclic AMP-dependent protein kinase, cyclic AMP and casein. The incorporation of ³²P into casein from [γ-³²P]ATP is measured by perchloric acid precipitation of the protein on filter paper. ³²P-Casein formation is linearly related to the specific radioactivity of the [γ-³²P]ATP. Separation of ATP from other ³²P-labeled compounds is not required for the assay. Application of this method in the evaluation of [γ-³²P]ATP specific radioactivity in two rat cardiac muscle preparations exposed to ³²P_i is demonstrated.