cyclic-gmp and diadenosine-tetraphosphate

Topics: Adenosine Triphosphate; Amino Acids; Binding Sites; Cyclic AMP; Cyclic GMP; Dinucleoside Phosphates; Humans; Hydrolysis; Phosphoric Diester Hydrolases; Protein Binding; Pyrophosphatases; Substrate Specificity; Thymidine Monophosphate; Uridine Triphosphate

Dinucleoside tetraphosphates are common constituents of the cell and are thought to play diverse biological roles in organisms ranging from bacteria to humans. In this study we characterized two independent mechanisms by which di-adenosine tetraphosphate (Ap4A) metabolism impacts biofilm formation by Pseudomonas fluorescens. Null mutations in apaH, the gene encoding nucleoside tetraphosphate hydrolase, resulted in a marked increase in the cellular level of Ap4A. Concomitant with this increase, Pho regulon activation in low-inorganic-phosphate (P(i)) conditions was severely compromised. As a consequence, an apaH mutant was not sensitive to Pho regulon-dependent inhibition of biofilm formation. In addition, we characterized a Pho-independent role for Ap4A metabolism in regulation of biofilm formation. In P(i)-replete conditions Ap4A metabolism was found to impact expression and localization of LapA, the major adhesin regulating surface commitment by P. fluorescens. Increases in the level of c-di-GMP in the apaH mutant provided a likely explanation for increased localization of LapA to the outer membrane in response to elevated Ap4A concentrations. Increased levels of c-di-GMP in the apaH mutant were associated with increases in the level of GTP, suggesting that elevated levels of Ap4A may promote de novo purine biosynthesis. In support of this suggestion, supplementation with adenine could partially suppress the biofilm and c-di-GMP phenotypes of the apaH mutant. We hypothesize that changes in the substrate (GTP) concentration mediated by altered flux through nucleotide biosynthetic pathways may be a significant point of regulation for c-di-GMP biosynthesis and regulation of biofilm formation.

Topics: Bacterial Proteins; Biofilms; Cloning, Molecular; Cyclic GMP; Dinucleoside Phosphates; Gene Expression Regulation, Bacterial; Mutation; Oxidative Stress; Pseudomonas fluorescens; Purines

By use of front-surface fluorometry and fura-2-loaded medial strips of the porcine coronary artery, cytosolic Ca++ concentration ([Ca++]i) and force development were monitored simultaneously to determine the mechanisms of vasorelaxation induced by the diadenosine polyphosphates (APnA) diadenosine 5',5'''-P1, P4-tetraphosphate (AP4A) and diadenosine 5',5'''-P1,P5-pentaphosphate (AP5A). APnA concentration-dependently inhibited the sustained elevations of [Ca++]i and force induced by U-46619, a thromboxane A2 analog, in the presence of extracellular Ca++. APnA shifted the [Ca++]i-force relation curves of contractions induced by various concentrations of high K+ to the right. The AP4A-induced decreases in [Ca++]i and force were largely attenuated by tetrabutylammonium. The AP4A-induced decreases in force were attenuated by 4-aminopyridine and charybdotoxin. The AP5A-induced decreases in [Ca++]i and force were attenuated by tetrabutylammonium, 4-aminopyridine and charybdotoxin. In the absence of extracellular Ca++, APnA did not inhibit the transient elevations of [Ca++]i induced by histamine or caffeine. Both AP4A and AP5A increased intracellular cAMP content. We thus conclude that AP4A and AP5A relax the porcine coronary artery by decreasing [Ca++]i, possibly through the activation of K+ channels, but not through inhibition of intracellular Ca++ release and by decreasing the Ca++ sensitivity of the contractile machinery. These effects were considered to be mediated by cAMP.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Caffeine; Calcium; Coronary Vessels; Cyclic AMP; Cyclic GMP; Dinucleoside Phosphates; Histamine; In Vitro Techniques; Muscle, Smooth, Vascular; Potassium Channel Blockers; Swine; Vasodilation

Di(adenosine-5')oligophosphate nucleotides of general structure ApnA (n = 2-6) inhibited phosphorylation of immunoglobulin G from tumor-bearing rabbits (TBR IgG) by pp60src protein kinase purified from Rous sarcoma virus-transformed rat tumor cells. Ap4A, a nucleotide associated with eukaryotic cell proliferation, was one of the most effective inhibitors in the series, causing 50% inhibition of TBR IgG phosphorylation at 15 microM. Ap4A inhibited pp60src-dependent phosphorylation of TBR IgG in solution and immunoprecipitates, as well as the phosphorylation of tubulin, microtubule-associated proteins, and vinculin. Under similar assay conditions, Ap4A did not inhibit phosphorylation of histone H2b by cAMP- or cGMP-dependent protein kinases. Ap4A appears to interact noncovalently with the enzyme, because removal of pp60src by immunoprecipitation from solutions containing Ap4A restored activity to uninhibited levels. A 100-fold increase in ATP (4-400 nM) caused a 13-fold increase in the 50% inhibitory concentration of Ap4A (2.5-33 microM), consistent with the interpretation that Ap4A competes for an ATP-binding site on the pp60src molecule. The simplest explanation of these results is that Ap4A binds to the phosphodonor site for ATP.

Topics: Adenine Nucleotides; Animals; Cell Line; Cell Transformation, Neoplastic; Cyclic AMP; Cyclic GMP; Dinucleoside Phosphates; Immunoglobulin G; Kinetics; Neoplasms, Experimental; Oncogene Protein pp60(v-src); Phosphorylation; Protein Kinases; Rabbits; Rats; Viral Proteins