The effect of piroxicam on platelet aggregation

Summary Piroxicam inhibited aggregation of human and dog platelets caused by collagen, but not by adenosine diphosphate (ADP). Release of platelet ADP was inhibited by piroxicam.     

Effects of methoprene and juvenile hormone on the oxidative metabolism of isolated mitochondria from flight muscle ofLocusta migratoria L.

Summary In vitro applications of juvenile hormone III and a juvenile hormone analogue, methoprene, were made to mitochondria isolated from dorsal longitudinal flight muscles of adultLocusta migratoria L. Both compounds completely inhibited oxygen consumption at the highest concentrations used. At lower concentrations, state 3 respiration and respiratory control were reduced but the ADP/O ratio was largely unaffected.     

Inhibition of rat liver transglutaminase by nucleotides.

Tissue-type transglutaminase (TGase) was purified from rat liver, and the effects of nucleotides on its activity were examined. The enzyme activity is inhibited by ATP in a concentration-dependent way, with complete inhibition by 3 mM ATP. Partially-purified TGase from human brain was inhibited by ATP in a manner similar to that observed with the rat liver enzyme. This suggests that the inhibition is a common phenomenon for tissue-type TGase in all species and tissues. The inhibition is reversible since full activity is restored by lowering the ATP concentration. CTP has a TGase-inhibitory potency equivalent to that of ATP, whereas GTP and UTP possess about 50% of the inhibitory activity of ATP. ADP inhibits TGase activity to the same extent as ATP, but AMP causes much less inhibition, and there is no inhibition by adenosine or adenine. The inhibition by ATP is insensitive to ionic strength and is non-competitive with the substrate putrescine. Since ATP levels in cells are of mM order, these results suggest that TGase activity is controlled by ATP in vivo.     

Inhibition of rat liver transglutaminase by nucleotides

Summary Tissue-type transglutaminase (TGase) was purified from rat liver, and the effects of nucleotides on its activity were examined. The enzyme activity is inhibited by ATP in a concentration-dependent way, with complete inhibition by 3 mM ATP. Partially-purified TGase from human brain was inhibited by ATP in a manner similar to that observed with the rat liver enzyme. This suggests that the inhibition is a common phenomenon for tissue-type TGase in all species and tissues. The inhibition is reversible since full activity is restored by lowering the ATP concentration. CTP has a TGase-inhibitory potency equivalent to that of ATP, whereas GTP and UTP possess about 50% of the inhibitory activity of ATP. ADP inhibits TGase activity to the same extent as ATP, but AMP causes much less inhibition, and there is no inhibition by adenosine or adenine. The inhibition by ATP is insensitive to ionic strength and is non-competitive with the substrate putrescine. Since ATP levels in cells are of mM order, these results suggest that TGase activity is controlled by ATP in vivo.     

Platelet aggregation is inhibited by phycolectins

Lectins from four marine algal species were examined for interaction with human platelets. The lectin designated hypnin A, from the red algaHypnea japonica, inhibited adenosine diphosphate (ADP)-or collagen-induced human platelet aggregation in a dose-dependent manner. Complete inhibition was observed at concentrations of 100 and 5 g/ml of the lectin with, ADP (2 M) and collagen (0.2 g/ml)-induced platelet aggregation, respectively. At the inhibitory concentration of 0.5 to 100 g/ml, the lectin did not induce aggregation of resting platelets. Lectins from the other three algal species also inhibited ADP-induced human platelet aggregation. These results indicate that the algal lectins are a new group of inhibitors and may be useful to study glycoconjugates on platelet membranes and to design novel platelet aggregation inhibitors.     

Participation of granulocytes and humoral factors in resolution of platelet aggregates during endotoxemia

Summary Hydrogen peroxide generated by phagocytizing granulocytes can prevent platelet aggregation induced by ADP or collagen but not by endotoxin. Endotoxin tolerance enhances granulocyte mobilization in response to endotoxin and reduces aggregation induced by endotoxin but not ADP or collagen.     

The involvement of fructose 2,6-bisphosphate in substrate cycle control in the nonoxidative stage of the pentose phosphate pathway. A phosphorus magnetic resonance spectroscopy study

The role of fructose 2,6-bisphosphate in the interconversion of sedoheptulose 7-phosphate and sedoheptulose 1,7-bisphosphate in rat liver cytosol fractions was studied by means of phosphorus magnetic resonance spectroscopy. When the activit of 6-phosphofructo-1-kinase was inhibited by a high concentration of ATP, the addition of fructose 2,6-bisphosphate led to a marked decrease in sedopheptulsoe 7-phosphate levels, accompanied by an increased concentration of ADP. Frructose 2,6-bisphosphate essentially inhibited both the decrease in sedoheptulose 1,7-disphosphate concentration and the accumulation of P_i in the incubation mixture. The data provided evidence that fructose 2,6-bisphosphate can regulate the substrate cycle; sedoheptulose 7-phosphate sedoheptulose 1,7-bisphosphate in the liver, and thus control the flux through the nonoxidative stage of the pentose phosphate pathway.     

Analysis of nucleotides bound to the ATP synthetase from spinach chloroplasts isolated under different conditions

Summary The total amount of bound adenine nucleotides in the coupling factor isolated from spinach chloroplasts and its distribution on AMP, ADP and ATP was analyzed after various incubation conditions. During purification of the coupling factor, the distribution of AMP, ADP and ATP is not altered. The coupling factor from deenergized membranes contains approximately 1 ADP, less than 1 ATP, and small amounts of AMP. During phosphorylation the pattern is changed and ATP becomes the dominant species. When exogenous ADP is lacking, phosphate is readily incorporated into ATP. Inhibition of adenylate kinase by AP₅A does not change the distribution pattern of the adenine nucleotides. The distribution pattern shows no integer numbers for the different nucleotides, suggesting that the coupling factor is present in different states in a statistical distribution.Acknowledgment: We thank the Swiss National Foundation for Scientific Research (grant 3.582.79) for generous support.     

Myocardial concentrations of high energy phosphates in normal mini-pigs and dogs

Zusammenfassung Die Gewebskonzentrationen an ATP, ADP, ATP+ADP, Kreatinphosphat, Kreatin und Kreatinphosphat + Kreatin sind unter vergleichbaren Bedingungen im linken Ventrikel von Mini-pig-Herzen kleiner als im Hundeherzen. Die Unterschiede in den Koeffizienten (ATP/ADP und Kreatinphosphat/Kreatin) dürfen narkosebedingt sein. Sämtliche Messgrössen sind nicht mit dem Entnahmeort der Biopsien (apikal, basal) korreliert.

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Supported by the Swiss National Science Foundation (Grant Nr. 3.484).     

Cell surface adenylate kinase activity regulates the F1-ATPase/P2Y13-mediated HDL endocytosis pathway on human hepatocytes

We have previously demonstrated on human hepatocytes that apolipoprotein A-I binding to an ecto-F₁-ATPase stimulates the production of extracellular ADP that activates a P2Y₁₃-mediated high-density lipoprotein (HDL) endocytosis pathway. Therefore, we investigated the mechanisms controlling the extracellular ATP/ADP level in hepatic cell lines and primary cultures to determine their impact on HDL endocytosis. Here we show that addition of ADP to the cell culture medium induced extracellular ATP production that was due to adenylate kinase and nucleoside diphosphokinase activities, but not to ATP synthase activity. We further observed that in vitro modulation of both ecto-NDPK and AK activities could regulate the ADP-dependent HDL endocytosis. But interestingly, only AK appeared to naturally participate in the pathway by consuming the ADP generated by the ecto-F₁-ATPase. Thus controlling the extracellular ADP level is a potential target for reverse cholesterol transport regulation. Received 13 July 2006; received after revision 29 August 2006; accepted 19 September 2006     

Inhibition of platelet ADP and serotonin release by carbon monoxide and in cigarette smokers

Summary The release of¹⁴C-serotonin by ADP, epinephrine and arachidonic acid and the release of ADP by kaolin were measured in normal platelets in the presence and absence of carbon monoxide and in smokers' platelets. It is shown that carbon monoxide inhibits significantly the platelet release reaction. This function is also decreased in platelets obtained from heavy cigarette smokers.This work was supported by Veterans Administration Research Fund 8073-01.     

Multiple flavonoid-binding sites within multidrug resistance protein MRP1

Recombinant nucleotide-binding domains (NBDs) from human multidrug resistance protein MRP1 were overexpressed in bacteria and purified to measure their direct interaction with high-affinity flavonoids, and to evaluate a potential correlation with inhibition of MRP1-mediated transport activity and reversion of cellular multidrug resistance. Among different classes of flavonoids, dehydrosilybin exhibited the highest affinity for both NBDs, the binding to N-terminal NBD1 being prevented by ATP. Dehydrosilybin increased vanadate-induced 8-N₃-[-³²P]ADP trapping, indicating stimulation of ATPase activity. In contrast, dehydrosilybin strongly inhibited leukotriene C₄ (LTC₄) transport by membrane vesicles from MRP1-transfected cells, independently of reduced glutathione, and chemosensitized cell growth to vincristine. Hydrophobic C-isoprenylation of dehydrosilybin increased the binding affinity for NBD1, but outsite the ATP site, lowered the increase in vanadate-induced 8-N₃-[-³²P]ADP trapping, weakened inhibition of LTC₄ transport which became glutathione dependent, and induced some cross-resistance. The overall results indicate multiple binding sites for dehydrosilybin and its derivatives, on both cytosolic and transmembrane domains of MRP1.Received 1 May 2003; received after revision 18 June 2003; accepted 24 June 2003     

The turnover of F-actin-bound ADP in vivo.

A procedure for estimating the rate of turnover of F-actin-bound ADP in vivo is described. A turnover rate of 0.88 h-1 was determined for mouse muscle F-actin. The validity of the method when used to estimate the turnover rate of F-actin per se is discussed in relation to the possible exchange of F-actin-bound ADP.     

ADP receptor P2Y13 induce apoptosis in pancreatic β-cells

Pancreatic β-cell loss represents a key factor in the pathogenesis of diabetes. Since the influence of purinergic signaling in β-cell apoptosis has not been much investigated, we examined the role of the ADP receptor P2Y₁₃ using the pancreatic insulinoma-cell line MIN6c4 as a model system. Real time-PCR revealed high expression of the ADP receptors P2Y₁ and P2Y₁₃. Adding the ADP analogue, 2MeSADP, to MIN6c4 cells induced calcium influx/mobilization and inhibition of cAMP production by activation of P2Y₁ and P2Y₁₃, respectively. 2MeSADP reduced cell proliferation and increased Caspase-3 activity; both these effects could be fully reversed by the P2Y₁₃ receptor antagonist MRS2211. We further discovered that blocking the P2Y₁₃ receptor results in enhanced ERK1/2, Akt/PKB and CREB phosphorylation mechanisms involved in β-cell survival. These results indicate that P2Y₁₃ is a proapoptotic receptor in β-cells as the P2Y₁₃ receptor antagonist MRS2211 is able to protect the cells from ADP induced apoptosis.     

Altersbedingte Abnahme von Kreatinphosphat und Änderungen der Adeninnukleotide in der Skelettmuskulatur von Ratten

Summary The content of phosphocreatine and of the adenin nucleotides, ATP, ADP and AMP in the white skeletal muscle of rats of different ages has been determined.There is an age-dependent relation between the quantity of phosphocreatine and the ratio of ATP/ADP. Young rats contain relatively much phosphocreatine (up to 57%) and a high ATP/ADP ratio (mean 6.7) while old rats show less phosphocreatine (40.8%) and also a low ATP/ADP ratio (mean 2.2).

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M. Ermini dankt der Schweizerischen Akademie der Medizinischen Wissenschaften für eine Verlängerung seines Stipendiums undI. Szelenyi dankt für ein USA Forschungs-Stipendium an das Institut für experimentelle Gerontologie in Basel. Herrn Prof.F. Verzár sind wir für die Leitung dieser und weiterer Arbeiten zu Dank verbunden.     

The possible role of isoforms of cytochrome c oxidase subunit VIa in mammalian thermogenesis

A single cDNA of cytochrome c oxidase subunit VIa was characterised from liver, heart and the thermogenic organ of the partially endotherm tuna fish. The amino acid sequence revealed high identity with subunit VIa from carp and trout, but low identity to subunits VIaL (liver type) and VIaH (heart type) of mammalian cytochrome c oxidase. In reconstituted cytochrome c oxidase from bovine heart, the H ⁺/e⁻ stoichiometry is decreased from 1.0 to 0.5 at high intraliposomal ATP/ADP ratios via exchange of bound ADP by ATP at the matrix domain of the transmembraneous subunit VIaH. Reconstituted cytochrome c oxidase from bovine liver and kidney, containing subunit VIaL, revealed H ⁺/e⁻ ratios below 0.5, independent of the ATP/ADP ratio. The results suggest the evolution of three types of subunit VIa. Subunits VIaH and VIaL are postulated to participate in mammalian thermogenesis. Received 3 May 1999; received after revision 10 June 1999; accepted 29 June 1999     

Mn2+ electron spin resonance studies on ATP phosphoribosyltransferase fromE. coli

Summary ESR binding studies of Mn²⁺ with each of the substrates and products suggests that substrate bridge complexes are formed in the reaction. This prediction is confirmed, comparing Mn²⁺+ADP and Mn²⁺+ADP+enzyme spectra.We thank V. M. Fernández for helpful discussions.     

The turnover of F-actin-bound ADP in vivo

Summary A procedure for estimating the rate of turnover of F-actin-bound ADP in vivo is described. A turnover rate of 0.88 h^–1 was determined for mouse muscle F-actin. The validity of the method when used to estimate the turnover rate of F-actin per se is discussed in relation to the possible exchange of F-actin-bound ADP.Acknowledgments. The invaluable technical assistance of Mr L. Carrington is gratefully acknowledged.     

The effect of myokinase on the aggregation and disaggregation of thrombocytes

Zusammenfassung Myokinase, welche die Umwandlung von 2 Molekülen ADP in je 1 Molekül AMP und ATP katalysiert, hemmt die durch ADP sowie durch Adrenalin hervorgerufene Thrombozytenaggregation beträchtlich. Myokinase unterstützt die Desaggregation der durch Adrenalin aggregierten Thrombozyten. Diese Befunde weisen darauf hin, dass die makroergen Phosphate nicht nur für die Aggregation, sondern auch für die Desaggregation eine bedeutende Rolle spielen.     

Phosphatases ofMycobacterium 607

Zusammenfassung Die Phosphatase(n)-Aktivität der zellfreien Extrakte vonMycobacterium 607 wurde untersucht und Pyrophosphat, ATP, ADP und Fructose-1,6-diphosphat hydrolisiert. Eigene Eigenschaften der Phosphatasen wurden studiert. Auch wurde nachgewiesen, dass die Hydrolyse von ATP und ADP durch mindestens zwei verschiedene Enzyme hervorzurufen ist.

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Supported in part by funds from Indian Council of Medical Research, New Delhi and Grant No. E-3427, National Institute of Allergy and Infectious Diseases U.S.P.H.S. Thanks are due to Dr.R. Viswanathan for his interest in this work.