Adaling Ogilvie

Human monocyte derived dendritic cells express functional P2X and P2Y receptors as well as ecto-nucleotidases

We investigated the expression and function of P2 receptors and ecto‐nucleotidases on human monocyte derived dendritic cells (DC). In addition we analyzed the effect of extracellular ATP on the maturation of DC. By RT‐PCR, DC were found to express mRNA for several P2X (P2X1, P2X4, P2X5, P2X7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y5, P2Y6, P2Y10, P2Y11) receptors. As shown by FURA‐2 measurement, triggering of P2 receptors resulted in an increase in free intracellular Ca2+. In combination with Tumor necrosis factor‐α, ATP increased the expression of the DC surface markers CD80, CD83 and CD86 indicating a maturation promoting effect. DC expressed the ecto‐apyrase CD39 and the ecto‐5′‐nucleotidase CD73 as demonstrated by RT‐PCR. Extracellular ATP was rapidly hydrolyzed by these ecto‐enzymes as shown by separation of 3H‐labeled ATP metabolites using a thin layer technique. These data suggest that ATP acts as a costimulatory factor on DC maturation.

The presence of diadenosine 5′,5‴-P1,P3-triphosphate (Ap3A) in human platelets

Diadenosine triphosphate (Ap3A) has been identified and quantified in human platelets using a coupled enzymatic assay specific for Ap3A, after fractionation of acidic extracts with high-performance liquid chromatography. Upon thrombin-induced aggregation, Ap3A is released together with the homologue diadenosine tetraphosphate (Ap4A). Extracts of human platelets do also contain enzymatic activities that degrade diadenosine tetraphosphate as well as diadenosine triphosphate. These enzymes, however, are not released during thrombin-induced aggregation of the platelets.

Diadenosine triphosphate (Ap3A) mediates human platelet aggregation by liberation of ADP

Human platelets store considerable amounts of diadenosine 51,5111-P1, P3-triphosphate, which is released together with the homologue diadenosine tetraphosphate (Ap4A) upon thrombin-induced aggregation (Lüthje,J. & Ogilvie,A. (1983) Biochem. Biophys. Res. Commun. 115, 253-260). We now report that, when added to platelet-rich plasma at 10-20 micron, diadenosine triphosphate gradually induces aggregation. The addition of diadenosine tetraphosphate antagonizes this effect by rapidly disaggregating the platelets. When another physiological but structurally unrelated stimulus, i.e. PAF (Platelet activating factor) is introduced into the system, diadenosine triphosphate drastically enhances and prolongs the aggregatory effect of PAF. Again, Ap4A is antagonistic in this system. The mechanism of Ap3A-stimulation can be explained by the slow and continuous liberation of ADP from Ap3A by the action of a hydrolyzing enzyme which is present in human plasma. Our studies suggest that Ap3A may be physiologically important in providing a relatively long-lived stimulus that can modulate platelet aggregation.

Compartmentalization of polyamines in mammalian cells.

A rapid method for the separation of cytoplasm and nuclei has been adapted for polyamine analysis of the different compartments of BSC-1 cells. The whole cells contain putrescine, spermidine and spermine at concentrations of 7.3, 6.5, and 12.4 nmol/106 cells respectively. By separating the cytoplasm and the nuclei within 20 seconds we found strikingly different polyamine distributions in the cell compartments. In the cytoplasm putrescine and spermine are present in almost equal amounts, whereas in the nucleus spermine is in 5-fold excess over putrescine and spermidine. Fluctuations of polyamines in the different cell compartments were investigated during the cell cycle after synchronizing the cells with thymidine/hydroxyurea treatment. An increase of putrescine in the cytoplasm parallels the initiation of DNA synthesis.

Vasoactive Diadenosine Polyphosphates Promote Growth of Cultured Renal Mesangial Cells

Diadenosine polyphosphates (diadenosine triphosphate, Ap3A; diadenosine tetraphosphate, Ap4A; diadenosine pentaphosphate, Ap5A; diadenosine hexaphosphate, Ap6A) are potent vasoactive molecules stored and released by platelets. We examined whether these dinucleotides might contribute to the glomerular inflammatory response by stimulating the proliferation of mesangial cells. In cultured rat mesangial cells all four tested dinucleotides (10 to 100 mumol/L) significantly stimulated DNA synthesis as measured by [3H]thymidine uptake at 48 hours (x-fold increase compared with unstimulated control cells: Ap3A, 1.5; Ap4A, 1.8; Ap5A, 1.6; Ap6A, 1.6). In combination with the platelet products platelet-derived growth factor, epidermal growth factor, and serotonin, the dinucleotides synergistically increased DNA synthesis. Dinucleotides by themselves increased cell counts by 23% to 43% at day 2 and augmented mesangial cell growth induced by platelet-derived growth factor, epidermal growth factor, and serotonin. Furthermore, dinucleotides (100 mumol/L) rapidly induced a modest increase in expression of the early growth response gene Egr-1 at 30 minutes (x-fold increase over baseline control: Ap3A, 1.9; Ap4A, 2.8; Ap5A, 2.2; Ap6A, 2.1). We found that extracellular Ap4A was metabolized by mesangial cell ectoenzymes to mononucleotides and adenosine, which also have been shown to be mitogenic for mesangial cells. The combination of Ap4A with mononucleotides or adenosine failed to cause additive stimulation of DNA synthesis in mesangial cells. We conclude that diadenosine polyphosphates stimulate proliferation of cultured mesangial cells and augment mesangial cell growth induced by other mitogens released from platelets. Different molecular mechanisms may be involved in dinucleotide-induced mitogenesis of mesangial cells. Direct effects of dinucleotides on cultured mesangial cells. Direct effects of dinucleotides on cultured mesangial cells appear to play a role because dinucleotides rapidly caused activation of Egr-1.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel orphan G protein-coupled receptor primarily expressed in the brain is localized on human chromosomal band 2q21

Abstract: A human hippocampus cDNA library was screened with a probe obtained from degenerate RT‐PCR aimed at P2Y‐homologous sequences. A positive clone, designated hip4, was identified containing an open reading frame of 1,020 bp that had been previously detected in a published genomic clone called R12. Subsequent screening of a human fetal brain cDNA library yielded a splice variant with a 1,104‐bp open reading frame, which was named fb1. Both variants display the seven‐transmembrane topology that is typical for G protein‐coupled receptors. Probing a human multitissue northern blot revealed two brain‐specific transcripts of 2.3 and 6.3 kb, respectively. Northern blot analyses with specific fragments confirmed that the two transcripts are generated by alternative polyadenylation yielding two different 3′ untranslated regions. A genomic clone from the corresponding gene was isolated and mapped to human chromosomal band 2q21 by fluorescence in situ hybridization.

Effects of diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A) on platelet aggregation in unfractionated human blood

SummaryThe effects on platelet aggregation of diadenosine triphosphate (Ap3A) and diadenosine tetraphosphate (Ap4A), both of which are stored in and released from platelet granules, have been studied in unfractionated human blood using a microscopic platelet-count ratio method. Ap3A at submicromolar concentrations induces platelet aggregation whereas the homologue dinucleotide Ap4A has disaggregating potency. In the concentration range between 10−7 to 10−5 M, Ap3A has been found to be as effective as ADP in triggering aggregate formation. These results confirm and essentially extend our recent findings with platelet-rich plasma that Ap3A is able to trigger platelet aggregation by a slow release of ADP from Ap3A which is catalyzed by a plasma hydrolase. Formation of platelet aggregates was also followed kinetically using a turbidometric method which has been developed for this purpose. In contrast to ADP which very rapidly induces a transient state of aggregation, the effect of Ap3A occurs much more slowly but induces the same maximum of aggregation. The duration of the Ap3A stimulus, however, is longer than that of ADP pointing to a potential physiological function of Ap3A as a “masked” source for ADP.

Biphasic cytotoxic mechanism of extracellular ATP on U-937 human histiocytic leukemia cells: involvement of adenosine generation

Since extracellular ATP can exhibit cytotoxic activity in vivo and in vitro, its application has been proposed as an alternative anticancer therapy. In this study we investigated the mechanisms of ATP-induced cytotoxicity in a human leukemic cell line (U-937). ATP added as a single dose exceeding 50 microM was cytostatic or even cytotoxic for U-937 cells. Interestingly, growth inhibition by ATP (50-3500 microM) showed a biphasic dose response. Up to 800 microM, ATP was cytotoxic in a dose-dependent manner (EC(50) 90 microM). In a range between 800 and 2500 microM, cell count was markedly higher despite the higher ATP concentrations. The cytotoxic effect of ATP could be antagonized by addition of uridine as a pyrimidine source and, alternatively, by addition of the nucleoside transmembrane inhibitor dipyridamole. The apoptosis-inducing adenosine A(3) receptor was not involved in measurable quantities, since (1) adenosine did not lead to an elevation of intracellular calcium levels, and (2) an unselective A(1-3) antagonist (ULS-II-80) could not abrogate the cytotoxic effect. Experiments monitoring extracellular nucleotide metabolism confirmed the assumption that the long-term production and continuous uptake of adenosine, which is extracellularly generated by degradation of ATP, led to an intracellular nucleotide imbalance with pyrimidine starvation. The biphasic dose response to higher ATP concentrations could be explained by the rapid degradation of lower ATP concentrations (300 microM) to adenosine by serum-derived enzymes, whereas higher concentrations (900 microM) only produced small amounts of adenosine due to forward inhibition of AMP hydrolysis by prolonged high ADP levels. FACS analysis revealed that at lower adenosine concentrations (300 microM) a reversible G(1) phase arrest of the cell cycle was induced, whereas higher concentrations (1000 microM) triggered apoptosis. Considering ATP as a potential cytostatic drug, our data have important implications concerning metabolic interactions of administered nucleotides.

Control of protein synthesis in mammalian cells by aminoacylation of transfer ribonucleic acid

The dependence of protein synthesis on the intracellular content of aminoacylated tRNA has been studied in mouse ascites tumor cells deprived for various amino acids. A remarkable reduction in net protein synthesis has been found only after a drastic decrease in aminoacylation of tRNA. The quantitative correlation of protein synthesis with the degree of aminoacylation suggests that a moderate amino acid starvation primarily influences the rate of elongation at the codon concerned. These results are in contrast to the findings previously reported for HeLa cells. Some crucial steps during the determination of intracellular aminoacyl-tRNA have been investigated. The reliability of the method employed has been discussed on a theoretical basis.

Nucleotide metabolizing ectoenzymes are upregulated in A431 cells periodically treated with cytostatic ATP leading to partial resistance without preventing apoptosis.

Extracellular ATP, when added as a single dose at concentrations higher than 0.1 mM to the culture medium, was growth inhibitory or even cytotoxic for human epidermoid carcinoma cells (A431). Adenosine at the same concentrations was much less potent. The molecular mechanism underlying the inhibitory effect of extracellular ATP has been investigated. The cytostatic as well as the cytotoxic effects of ATP could be prevented by supplying uridine as a pyrimidine source and, alternatively, by simultaneous addition of dipyridamole, which inhibits the uptake of adenosine. The data suggest that the long-term production and continuous uptake of adenosine, which is enzymatically generated from the ATP in the medium, led to an intracellular nucleotide imbalance with pyrimidine starvation. This triggered suicidal processes ending up in apoptosis of the cells. The tumor cells have been adapted to extracellular ATP with the aim to obtain cells which are more resistant to ATP. Therefore, growing cells were periodically treated with extracellular ATP. These cells were characterized by an enlargement of cell size, a decreased proliferation rate, and a reduced but not abolished sensitivity to cytostatic and cytotoxic ATP doses. The calcium response of adapted cells was shortened. The nucleotide hydrolyzing ectoenzyme activities (ecto-ATPase, ecto-ADPase, ecto-AMPase, ecto-Ap4Aase) were simultaneously upregulated. All phenotypic alterations of the adapted cells disappeared after cultivation for several generations in the absence of extracellular ATP. Considering ATP as a potential chemotherapeutic agent the adaptive phenomena of treated cells might be important.