Determination of ATP and ADP in blood platelets: A modification of the firefly luciferase assay for plasma

Although the rapid and considerable membrane depolarization response which accompanies activation of the phagocyte NADPH oxidase is due to transmembrane electron fluxes, little is known about the involvement of reactive oxidant species (ROS) in the subsequent repolarization response. In the current study, we have investigated the effects of superoxide dismutase (SOD), catalase, methionine, and the myeloperoxidase (MPO) inhibitors, sodium azide and 4-aminobenzoyl hydrazide (ABAH), as well as those of H 2 O 2 and HOCl (both at 100 μM) on the alterations in membrane potential which accompany activation of human neutrophils with the chemoattractant, FMLP (1 μM), and on store-operated uptake of Ca 2+ . The generation of ROS by FMLP-activated neutrophils was monitored according to the magnitude of oxygen consumption and autoiodination, while spectrofluorimetric procedures were used to measure alterations in membrane o op pe en nU UP P ( (J Ju un ne e 2 20 00 07 7) ) potential and influx of Ca 2+ . Treatment of the cells with H 2 O 2 , and HOCl, significantly impeded membrane repolarization, while sodium azide, ABAH, methionine, and catalase exerted the opposite effects, potentiating both the rates and the magnitudes of membrane repolarization and store-operated uptake of Ca 2+ . These observations demonstrate that NADPH oxidase regulates neutrophil membrane potential and Ca 2+ influx not only via its electrogenic activity, but also as a consequence of the generation of ROS.

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“…immediately prior to, and at 3 and 5 min following exposure of the cells to the chemoattractant, using a luciferin/luciferase procedure [10].…”

Section: Cellular Atp Levelsmentioning

confidence: 99%

Reactive oxidants regulate membrane repolarization and store-operated uptake of calcium by formyl peptide-activated human neutrophils

Tintinger

Theron

Potjo

2007

Free Radical Biology and Medicine

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“…ATP is released from aggregating thrombocytes (Holmsen et al, 1972;Meyers et al, 1982), endothelium (Schwiebert et al, 2002), sympathetic nerve endings (Burnstock & Sneddon, 1985;Satchell, 2000), and ischemic muscle cells (Gordon, 1986). Extracellular ATP exerts potent and diverse effects on the cardiovascular system via activation of P2 receptors (Gordon, 1986;Ralevic & Burnstock, 1998).…”

Section: Introductionmentioning

confidence: 99%

ATP‐induced vasodilation in human skeletal muscle

Ginneken

Meijer

Verkaik

et al. 2004

British J Pharmacology

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1 The purine nucleotide adenosine-5 0 -triphosphate (ATP) exerts pronounced effects on the cardiovascular system. The mechanism of action of the vasodilator response to ATP in humans has not been elucidated yet. The proposed endothelium-derived relaxing factors (EDRFs) were studied in a series of experiments, using the perfused forearm technique. 2 Adenosine 5 0 -triphosphate (0.2, 0.6, 6 and 20 nmol dl À1 forearm volume min À1 ) evoked a dosedependent forearm vasodilator response, which could not be inhibited by separate infusion of the nonselective COX inhibitor indomethacin (5 mg dl À1 min À1 , n ¼ 10), the blocker of Na þ /K þ -ATPase ouabain (0.2 mg dl À1 min À1 , n ¼ 8), the blocker of K Ca channels tetraethylammonium chloride (TEA, 0.1 mg dl À1 min À1 , n ¼ 10), nor by the K ATP -channel blocker glibenclamide (2 mg dl À1 min À1 , n ¼ 10). All blockers, except glibenclamide, caused a significant increase in baseline vascular tone. The obtained results might be due to compensatory actions of unblocked EDRFs. Combined infusion of TEA, indomethacin and L-NMMA (n ¼ 6) significantly increased the baseline forearm vascular resistance. The ATP-induced relative decreases in forearm vascular resistance were 4875, 6773, 8872, and 9272% in the absence and 2377, 6274, 8972, and 9371% in the presence of the combination of TEA, indomethacin and L-NMMA (Po0.05, repeated-measures ANOVA, n ¼ 6). A similar inhibition was obtained for sodium nitroprusside (SNP, Po0.05 repeated-measures ANOVA, n ¼ 6), indicating a nonspecific interaction due to the blocker-induced vasoconstriction. 3 ATP-induced vasodilation in the human forearm cannot be inhibited by separate infusion of indomethacin, ouabain, glibenclamide or TEA, or by a combined infusion of TEA, indomethacin, and L-NMMA. Endothelium-independent mechanisms and involvement of unblocked EDRFs, such as CO, might play a role, and call for further studies.

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“…From these analyses we calculated that our ATP standard contained 19 % ADP and 6 % AMP, our ADP standard 16 % AMP, and our AMP standard 0.1 % ATP and 0.4 % ADP. Although high, these contaminations are not exceptional compared to other reported impurity levels of ATP and ADP standards (Holmsen et al, 1966(Holmsen et al, , 1972 Kimmich et al, 1975). Taking the impurities of our standards into account, the light emissions with ADP-and AMP-reagents were expressed as percentages of those expected from the assays with ATP-reagent, assuming complete conversion (Table 1).…”

mentioning

confidence: 61%

Improvements of luciferin-luciferase methodology for determination of adenylate energy charge ratio of marine samples

Klinken¹,

Skjoldal²

1983

Mar. Ecol. Prog. Ser.

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Application of adenylate energy charge (EC) measurements in marine ecological studies requires high accuracy and precision. We have examined the luciferinluciferase method for EC determination and suggest the following methodological improvements. Replacement of the commonly used potassium phosphate buffer with potassium chloride in the assay medium prevents problems due to precipitation of magnesium phosphates. Interference from ADP and AMP in crude firefly extract can be eliminated by their enzymatic decomposition with apyrase and AMP-deaminase. Impure nucleotide standards are perhaps more common than currently realized, and can lead to inaccurate estimates of EC. proposed as an index of the energetical state of natural microbial communities and applied to both water and sediment samples (e. g. Wiebe and Bancroft, 1975;Witzel, 1979;Christensen and Devol, 1980;Karl, 1980;Romano and Daumas, 1981). EC measurements have also been used in studies of various types of stress in marine invertebrates (Ivanovici, 1980; Haya and Waiwood, in press). The EC ratio is normally stabilized at about 0.9 and changes in EC associated with environmental and physiological changes are usually small (Atkinson, 1977;Karl, 1980). High accuracy and precision in measurements are therefore important requirements for future evaluations of the usefulness of the EC ratio for ecological applications. We have examined the luciferin-luciferase methodology for EC determination with regard to potential interferences and standardization requirements. The adenylate energy charge ratio (EC =[Fifty mg freeze-dried firefly lantern extract (FLE-50; Sigma) were rehydrated with 10 to 25 m1 distilled water and aged successively for 4 and 12 to 48 h at room temperature and refrigerated, respectively. The preparation was cleared by centrifugation and warmed to room temperature prior to use. Enzymatic treatment of this FLE was performed by incubating a 5 rnl subsample with additions of 250 p1 AMP-deaminase (Sigma, Grade IV) and 0.5 mg apyrase (ATP-and ADPase; Sigma, Grade I) for 4 h at room temperature. Adenine nucleotides (AN) in the FLE preparations were extracted by injecting 0.5 m1 FLE into 4.5 m1 boiling Tris-buffer/EDTA (Lundin and Thore, 1975). ATP was assayed as described by Skjoldal and Barkati (1982). AMP and/or ADP were phosphorylated to ATP prior to assay by incubating (30 "C, 30 min) sample portions with phospho(eno1)pyruvate (PEP), adenylate kinase (AK) and/or pyruvate kinase (PK) (madet, 1967). Two different sets of incubation reagents, based on either potassium phosphate (KP,) buffer or potassium chloride (KCl), were compared in one experiment. In all other experiments the KC1-reagents were used. The KP, incubation method followed with slight modifications that of Chapman et al. (1971). To 250 p1 subsamples were added 50 p1 MgC1, (25 mM) and 50 p1 reagent. The reagent for determination of ATP (ATP-reagent) was KP,-buffer (K2HP0,/KH2P0,, 75 mM, pH 7.5). The reagent for determination of ATP + ADP (ADP-reagent) contained KP,-buffer,...

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Determination of ATP and ADP in blood platelets: A modification of the firefly luciferase assay for plasma

Cited by 315 publications

References 6 publications

Reactive oxidants regulate membrane repolarization and store-operated uptake of calcium by formyl peptide-activated human neutrophils

Reactive oxidants regulate membrane repolarization and store-operated uptake of calcium by formyl peptide-activated human neutrophils

ATP‐induced vasodilation in human skeletal muscle

Improvements of luciferin-luciferase methodology for determination of adenylate energy charge ratio of marine samples

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