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Extracellular nucleotides stimulate receptor-mediated calcium mobilization and inositol phosphate production in human fibroblasts.

ABSTRACT: In human skin fibroblasts, low concentrations of extracellular ATP stimulated 45Ca2+ efflux from a slow-turnover intracellular pool, accompanied by inositol phosphate generation. These effects of ATP were not due to a generalized increase in plasma-membrane permeability. The EC50 (concn. giving 50% stimulation) for ATP was dependent on Ca2+ and Mg2+ concentrations in a manner which indicates that a form of ATP uncomplexed with bivalent cations is the active species. The rank order of potency of nucleotides was: ATP = UTP greater than adenosine 5'-[gamma-thio]triphosphate greater than ITP greater than ADP greater than UDP greater than other nucleoside triphosphates. Adenosine 5'-[alpha beta-methylene]triphosphate, adenosine 5'-[beta gamma-methylene]triphosphate and 2-methylthio-ATP were inactive. Thus the nucleotide specificity of this receptor is different from that of previously characterized P2 purinoceptors. Nucleotide-stimulated 45Ca2+ mobilization and inositol phosphate production were markedly inhibited by phorbol ester, and partially inhibited by pertussis-toxin pretreatment. These findings suggest that the coupling of nucleotide receptor to phospholipase C is mediated both by a pertussis-toxin-sensitive G-protein and by a pertussis-toxin-insensitive mechanism.

SUBMITTER: Fine J

PROVIDER: S-EPMC1133439 | biostudies-other | 1989 Oct

REPOSITORIES: biostudies-other

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Project description:The pathway for the synthesis of di-myo-inositol-phosphate (DIP) was recently elucidated on the basis of the detection of the relevant activities in cell extracts of Archaeoglobus fulgidus and structural characterization of products by nuclear magnetic resonance (NMR) (N. Borges, L. G. Gonçalves, M. V. Rodrigues, F. Siopa, R. Ventura, C. Maycock, P. Lamosa, and H. Santos, J. Bacteriol. 188:8128-8135, 2006). Here, a genomic approach was used to identify the genes involved in the synthesis of DIP. Cloning and expression in Escherichia coli of the putative genes for CTP:l-myo-inositol-1-phosphate cytidylyltransferase and DIPP (di-myo-inositol-1,3'-phosphate-1'-phosphate, a phosphorylated form of DIP) synthase from several (hyper)thermophiles (A. fulgidus, Pyrococcus furiosus, Thermococcus kodakaraensis, Aquifex aeolicus, and Rubrobacter xylanophilus) confirmed the presence of those activities in the gene products. The DIPP synthase activity was part of a bifunctional enzyme that catalyzed the condensation of CTP and l-myo-inositol-1-phosphate into CDP-l-myo-inositol, as well as the synthesis of DIPP from CDP-l-myo-inositol and l-myo-inositol-1-phosphate. The cytidylyltransferase was absolutely specific for CTP and l-myo-inositol-1-P; the DIPP synthase domain used only l-myo-inositol-1-phosphate as an alcohol acceptor, but CDP-glycerol, as well as CDP-l-myo-inositol and CDP-d-myo-inositol, were recognized as alcohol donors. Genome analysis showed homologous genes in all organisms known to accumulate DIP and for which genome sequences were available. In most cases, the two activities (l-myo-inositol-1-P cytidylyltransferase and DIPP synthase) were fused in a single gene product, but separate genes were predicted in Aeropyrum pernix, Thermotoga maritima, and Hyperthermus butylicus. Additionally, using l-myo-inositol-1-phosphate labeled on C-1 with carbon 13, the stereochemical configuration of all the metabolites involved in DIP synthesis was established by NMR analysis. The two inositol moieties in DIP had different stereochemical configurations, in contradiction of previous reports. The use of the designation di-myo-inositol-1,3'-phosphate is recommended to facilitate tracing individual carbon atoms through metabolic pathways.

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Extracellular nucleotides stimulate receptor-mediated calcium mobilization and inositol phosphate production in human fibroblasts.

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