Malondialdehyde (MDA) is a product of lipid peroxidation and prostaglandin biosynthesis. It is mutagenic and carcinogenic and the major adduct formed by reaction with DNA, a highly fluorescent pyrimidopurinone (M1-dG), has been detected in healthy human liver and leukocyte DNA. Analytical methods used so far for the detection of M1-dG have not been applied to a large number of individuals or variety of samples. Often, only a few microg of DNA from human tissues are available for analysis and a very sensitive assay is needed in order to detect background levels of M1-dG in very small amounts of DNA. In this paper, the development of an immunoslot blot (ISB) assay for the measurement of MI-dG in 1 microg of DNA is described. The limit of detection of the assay is 2.5 adducts per 10(8) bases. A series of human samples were analysed and levels of 5.6-9.5 (n = 8) and 3.1-64.3 (n = 42) of M1-dG per 10(8) normal bases were detected in white blood cell and gastric biopsy DNA, respectively. Results on four human samples were compared with those obtained using an HPLC/32P-post-labelling (HPLC/PPL) method previously developed and indicated a high correlation between M1-dG levels measured by the two assays. The advantages of ISB over other assays including HPLC/PPL, such as the possibility of analysing 1 microg DNA/sample and the fact that it is less time-consuming and laborious, means that it can be more easily used for routine analysis of a large number of samples in biomonitoring studies.
ATP and ADP, in concentrations ranging from 1-100 microM, increased the release of [3H]choline and [3H]phosphorylcholine (P-choline) from bovine aortic endothelial cells (BAEC) prelabelled with [3H]choline. This action was detectable within 5 minutes and was maintained for at least 40 minutes. ATP and ADP were equiactive, and their action was mimicked by their phosphorothioate analogs (ATP gamma S and ADP beta S) and adenosine 5'-(beta, gamma imido) triphosphate (APPNP), but not by AMP, adenosine, and adenosine 5'-(alpha, beta methylene)triphosphate (APCPP): these results are consistent with the involvement of P2Y receptors. ATP also induced an intracellular accumulation of [3H]choline: the intracellular level of [3H]choline was increased 30 seconds after ATP addition and remained elevated for a least 20 minutes. The action of ATP on the release of choline metabolites was reproduced by bradykinin (1 microM), the tumor promoter phorbol 12-myristate 13-acetate (PMA, 50 nM), and the calcium ionophore A23187 (0.5 microM). Down-regulation of protein kinase C, following a 24-hour exposure of endothelial cells to PMA, abolished the effects of PMA and ATP on the release of choline and P-choline, whereas the response to A23187 was maintained. These results suggest that in aortic endothelial cells, ATP produces a sustained activation of a phospholipase D hydrolyzing phosphatidylcholine. The resulting accumulation of phosphatidic acid might have an important role in the modulation of endothelial cell function by adenine nucleotides. Stimulation of phospholipase D appears to involve protein kinase C, activated following the release of diacylglycerol from phosphatidylinositol bisphosphate by a phospholipase C coupled to the P2Y receptors (Pirotton et al., 1987a).
ATP and ATP gamma S(10-100 microM) stimulated the release of prostacyclin (PGI2) from bovine aortic smooth muscle cells. This effect was reproduced by UTP, ITP and partially by GTP. ADP and ADP beta S, the P2X-selective agonist alpha, beta-methylene ATP (APCPP), AMP and adenosine were all inactive. This effect of ATP gamma S was not inhibited by Reactive Blue 2, an antagonist of P2Y receptors. The stimulation of PGI2 production in aortic smooth muscle cells by these nucleotides thus seems to involve receptors distinct from both P2X and P2Y subtypes, which are responsible for smooth muscle contraction and PGI2 release from endothelial cells, respectively.
The release of prostacyclin (PGI2) from bovine aortic endothelial cells stimulated by adenosine 5′‐triphosphate (ATP) was decreased by amiloride analogues bearing alkyl groups on the 5‐amino nitrogen atom, like 5‐(N‐ethyl‐N‐isopropyl)amiloride (EIPA), which are inhibitors of the Na+/H+ exchanger. Analogues substituted on a terminal guanidino nitrogen atom were not inhibitory. The release of PGI2 induced by ATP was not significantly depressed in a Na+‐poor medium or in a medium acidified to pH 6.9, two conditions known to inhibit the Na+/H+ exchanger. Cytoplasmic alkalinization by ammonium chloride did not suppress the inhibitory action of EIPA. By itself, ammonium chloride decreased the response of endothelial cells to ionophore A23187 and ATP, whereas sodium acetate had no effect. EIPA did not decrease the mobilization of free arachidonic acid induced by ATP. It inhibited the conversion of exogenous arachidonate into PGI2 and prostaglandin E2 (PGE2). Although the intracellular pH was not measured in this study, it seems unlikely that cytoplasmic alkalinization via the activation of the Na+/H+ exchanger plays a significant role in the stimulatory action of ATP on the release of PGI2 from endothelial cells. The inhibition of that release by EIPA and other amiloride analogues might involve a direct effect on cyclo‐oxygenase, although an action on the reacylation of free arachidonic acid cannot be excluded.
1 Pretreatment of bovine aortic endothelial cells with cycloheximide enhanced their capacity to release prostacyclin in response to adenosine 5'-triphosphate (ATP) and bradykinin. 2 The action of cycloheximide was time-dependent; it became detectable after a 1 h exposure to the cells and was maximal after 3 h. 3 Puromycin mimicked the effect of cycloheximide. For these two agents, the enhancement of prostacyclin release was obtained at concentrations producing a partial inhibition of protein synthesis. 4 Cycloheximide increased the mobilization of free arachidonic acid induced by ATP in bovine aortic endothelial cells. 5 In conclusion, the synthesis of new proteins is not involved in the stimulatory action of ATP and bradykinin on prostacyclin production by bovine aortic endothelial cells. Despite the short half-life of prostaglandin H synthase in endothelial cells, cycloheximide and puromycin enhanced the release of prostacyclin induced by agonists. Our data suggest that this release might be under the control of rapidly turning-over phospholipase inhibitory proteins.
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