ATPace™: injectable adenosine 5′-triphosphate

Carbon monoxide (CO) is produced endogenously by heme oxygenase (HO) enzymes. HO-1 is highly expressed in many inflammatory disease states, where it is broadly protective. The protective effects of HO-1 expression can be largely mimicked by the exogenous application of CO and CO-releasing molecules (CORMs). Despite a dearth of pharmacological tools for their study, molecular methodologies have identified P2X4 receptors as a potential anti-nociceptive drug target. P2X4 receptors are up-regulated in animal models of inflammatory pain, and their knock-down reduces pain behaviours. In these same animal models, HO-1 expression is anti-nociceptive, and we therefore investigated whether P2X4 was a target for CO and tricarbonyldichlororuthenium (II) dimer (CORM-2). Using conventional whole-cell and perforated-patch recordings of heterologously expressed human P2X4 receptors, we demonstrate that CORM-2, but not CO gas, is an inhibitor of these channels. We also investigated the role of soluble guanylate cyclase and mitochondria-derived reactive oxygen species using pharmacological inhibitors but found that they were largely unable to affect the ability of CORM-2 to inhibit P2X4 currents. A control breakdown product of CORM-2 was also without effect on P2X4. These results suggest that P2X4 receptors are not a molecular target of endogenous CO production and are, therefore, unlikely to be mediating the anti-nociceptive effects of HO-1 expression in inflammatory pain models. However, these results show that CORM-2 is an effective antagonist at human P2X4 receptors and represents a useful pharmacological tool for the study of these receptors given the current dearth of antagonists.     

ATPace™: injectable adenosine 5′-triphosphate

ATPace?, a novel injectable formulation of adenosine 5′-triphosphate (ATP), is developed by Cordex Pharma, Inc. (Cordex) as a diagnostic and therapeutic drug for the management of cardiac bradyarrhythmias. Extracellular ATP exerts multiple effects in various cell types by activating cell-surface receptors known as P2 receptors. In the heart, ATP suppresses the automaticity of cardiac pacemakers and atrioventricular (AV) nodal conduction via adenosine, the product of its degradation by ecto-enzymes, as well as by triggering a cardio-cardiac vagal reflex. ATP, given as a rapid intravenous bolus injection, has been used since the late 1940s as a highly effective and safe therapeutic agent for the acute termination of reentrant paroxysmal supraventricular tachycardia (PSVT) involving the AV node. In addition, preliminary studies have shown that ATP can also be used as a diagnostic agent for the identification of several cardiac disorders including sinus node dysfunction (sick sinus syndrome), dual AV nodal pathways, long QT syndrome, and bradycardic syncope. The US Food and Drug Administration has approved Cordex formulation for ATP as an Investigational New Drug and two pathways for its marketing approval; one therapeutic, i.e., acute termination of paroxysmal PSVT, and the other diagnostic, i.e., the identification of patients with bradycardic syncope who can benefit from pacemaker therapy. The scientific rationale for the development of ATPace? is discussed.     

Amino acid activation with adenosine 5′-phosphorimidazolide

Summary Amino acids are activated by reaction with adenosine 5-phosphorimidazolide in aqueous imidazole buffers. If adenosine 5-(O-methylphosphate), an analogue of the 3-terminus of t-RNA is present, 2(3)-O-aminoacyladenosine 5-(O-methylphosphate) is formed. Fifteen percent of this compound accumulated at pH 5.8, but less was formed at higher pHs. The highest efficiency of utilization of ImpA attained in our experiments was about 24%. Analogous reactions occured with several other amino acids, including a number that have functional side-chains.Abbreviations pA adenosine 5-monophosphate - MepA adenosine-5-(O-methylphosphate) - ImpA adenosine-5-phosphorimidazolide - A adenosine - MepA-ala 2(3)-O-alanyl-adenosine-5-(O-methylphosphate) - ala-N-pA adenylyl-(5 N)-alanine - ImH imidazole - DKP diketopiperazine     

Localization of adenosine 5′-phosphosulfate sulfotransferase in spinach leaves

Roots of spinach (Spinacia oleracea L.) seedlings contained only a very low activity of adenosine 5-phosphosulfate sulfotransferase compared to the cotyledons. Adenosine 5-phosphosulfate sulfotransferase activity increased about tenfold in cotyledons during greening. Preparation of organelle fractions from spinach leaves by a combination of differential and isopycnic density gradient centrifugation showed that adenosine 5-phosphosulfate sulfotransferase banded with NADP-glyceraldehyde-3-phosphate dehydrogenase, a marker enzyme for intact chloroplasts. In the fractions of peroxisomes, mitochondria and broken chloroplasts virtually no adenosine 5-phosphosulfate sulfotransferase activity was measured. Comparison with the chloroplast enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase indicates that in spinach, adenosine 5-phosphosulfate sulfotransferase is localized almost exclusively in the chloroplasts.Abbreviations APS Adenosine 5-phosphosulfate - APSSTase Adenosine 5-phosphosulfate sulfotransferase - BSA Bovine serum albumin - BRIJ58 Polyethylene glycolmonostearylether - DTE Dithioerythritol - DTT Dithiothreitol - EDTA Ethylenediaminetetraacetic acid - ME 2-Mercaptoethanol - NADP-GPD NADP-linked glyceraldehyde-3-phosphate dehydrogenase - PAPS Adenosine 3-phosphate 5-phosphate 5-phosphosulfate - POPOP 1,4 Di [2-(5-phenyloxazolyl)]-benzene - PPO 2,5-Diphenyloxazol The results presented in this paper are taken from the Ph. D. thesis of H.F.     

Coordination of magnesium with adenosine 5′-diphosphate and triphosphate

³¹P NMR chemical shifts of salts of adenosine 5′-triphosphate and diphosphate: ATPH^2?₂2(Me₄N⁺) · H₂O, ATPH^2?₂2 Na⁺ · 3.5 H₂O, ATPH^2?₂Mg²⁺ · 4 H₂O, ATPH^2?₂Ca²⁺ · 2 H₂O, ADPH^2?2(Me₄N⁺) · H₂O and ADPH^2?Mg²⁺ · 4 H₂O have been measured in 0.02 M ²H₂O solutions at 145.7 MHz (22° C) at constant p²H values (8.20 and 6.20). The results are compared with those obtained from salts of adenosine 5′-monophosphate and other simpler phosphomonoesters, e.g. AMP^2?2(Me₄N⁺), AMP^2?Mg²⁺, AMPH^?Me₄N⁺ and (AMPH^?)₂Mg²⁺. It is concluded that the effects exerted by Mg²⁺ and Ca²⁺ on the ³¹P NMR shifts of dipoly- and tripolyphosphates relative to monovalent cations are due mainly to changes in conformation of the polyphosphate chain rather than to purely electronic factors associated with the binding of divalent cations to the phospho-oxyanions. The data are consistent with the existence of the following complexes at p²H 8.20: (MgP_αP_β)ADP^? and (MgP_αP_γ)ATP^2?af (MgP_αP_β)ATP^2?af (MgP_βP_γ)ATP^2? with the latter equilibrium relatively fast in the NMR time scale. Monoprotonation of the terminal phosphate appears to weaken the Mg²⁺-polyphosphate binding, particularly at P_β of MgADPH and at P_β and P_γ of MgATPH^?. The Mg²⁺-polyphosphate binding weakens further at p²H 3.70, i.e. in MgATPH₂. Possible implications of the results in the mechanism of actomyosin Mg²⁺-ATPase in muscle contraction are discussed.     

The ecto-enzymes CD73 and adenosine deaminase modulate 5′-AMP-derived adenosine in myofibroblasts of the rat small intestine

Adenosine is a versatile signaling molecule recognized to physiologically influence gut motor functions. Both the duration and magnitude of adenosine signaling in enteric neuromuscular function depend on its availability, which is regulated by the ecto-enzymes ecto-5′-nucleotidase (CD73), alkaline phosphatase (AP), and ecto-adenosine deaminase (ADA) and by dipyridamole-sensitive equilibrative transporters (ENTs). Our purpose was to assess the involvement of CD73, APs, ecto-ADA in the formation of AMP-derived adenosine in primary cultures of ileal myofibroblasts (IMFs). IMFs were isolated from rat ileum longitudinal muscle segments by means of primary explant technique and identified by immunofluorescence staining for vimentin and α-smooth muscle actin. IMFs confluent monolayers were exposed to exogenous 5′-AMP in the presence or absence of CD73, APs, ecto-ADA, or ENTs inhibitors. The formation of adenosine and its metabolites in the IMFs medium was monitored by high-performance liquid chromatography. The distribution of CD73 and ADA in IMFs was detected by confocal immunocytochemistry and qRT-PCR. Exogenous 5′-AMP was rapidly cleared being almost undetectable after 60-min incubation, while adenosine levels significantly increased. Treatment of IMFs with CD73 inhibitors markedly reduced 5′-AMP clearance whereas ADA blockade or inhibition of both ADA and ENTs prevented adenosine catabolism. By contrast, inhibition of APs did not affect 5′-AMP metabolism. Immunofluorescence staining and qRT-PCR analysis confirmed the expression of CD73 and ADA in IMFs. Overall, our data show that in IMFs an extracellular AMP-adenosine pathway is functionally active and among the different enzymatic pathways regulating extracellular adenosine levels, CD73 and ecto-ADA represent the critical catabolic pathway.     

The preparation of adenosine 5′-pyrophosphate by a non-enzymic method

1. A non-enzymic method for the preparation of adenosine 5′-diphosphate is described, in which the terminal phosphate of adenosine 5′-triphosphate is transferred to methanol in the presence of hydrochloric acid. The final purified product can be obtained in 60% yield. 2. Experiments with [¹⁴C]methanol showed that no methylation of the adenosine diphosphate occurs during the reaction. 3. Confirmation that the pyrophosphate moiety of the adenosine diphosphate produced was in the 5′-position was obtained by: (a) periodate oxidation; (b) treatment with apyrase and examination of the resulting adenylic acid isomer by paper chromatography. 4. The method appears to be generally applicable to the preparation of nucleoside 5′-diphosphates from the corresponding nucleoside 5′-triphosphates.     

Occurrence of adenosine 2′,5′-bisphosphate in rat liver

Adenosine 2′,5′-bisphosphate (pAp) is present in liver from 2-day-fasted rats, at a concentration of around 1 μM. pAp was obtained through perchloric acid extraction of the liver followed by two successive DEAE-cellulose chromatographies and an ion-pair high-pressure liquid chromatography. Both pAp extracted from liver and that obtained from a commercial source showed the same pattern of hydrolysis by alkaline phosphatase, i.e., more 5′-AMP than 2′-AMP was obtained as an intermediate of the reaction.     

Purification and properties of adenylyl sulphate:ammonia adenylyltransferase from Chlorella catalysing the formation of adenosine 5′-phosphoramidate from adenosine 5′-phosphosulphate and ammonia

Extracts of Chlorella pyrenoidosa, Euglena gracilis var. bacillaris, spinach, barley, Dictyostelium discoideum and Escherichia coli form an unknown compound enzymically from adenosine 5′-phosphosulphate in the presence of ammonia. This unknown compound shares the following properties with adenosine 5′-phosphoramidate: molar proportions of constituent parts (1 adenine:1 ribose:1 phosphate:1 ammonia released at low pH), co-electrophoresis in all buffers tested including borate, formation of AMP at low pH through release of ammonia, mass and i.r. spectra and conversion into 5′-AMP by phosphodiesterase. This unknown compound therefore appears to be identical with adenosine 5′-phosphoramidate. The enzyme that catalyses the formation of adenosine 5′-phosphoramidate from ammonia and adenosine 5′-phosphosulphate was purified 1800-fold (to homogeneity) from Chlorella by using (NH₄)₂SO₄ precipitation and DEAE-cellulose, Sephadex and Reactive Blue 2–agarose chromatography. The purified enzyme shows one band of protein, coincident with activity, at a position corresponding to 60000–65000 molecular weight, on polyacrylamide-gel electrophoresis, and yields three subunits on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of 26000, 21000 and 17000 molecular weight, consistent with a molecular weight of 64000 for the native enzyme. Isoelectrofocusing yields one band of pI4.2. The pH optimum of the enzyme-catalysed reaction is 8.8. ATP, ADP or adenosine 3′-phosphate 5′-phosphosulphate will not replace adenosine 5′-phosphosulphate, and the apparent K_m for the last-mentioned compound is 0.82mm. The apparent K_m for ammonia (assuming NH₃ to be the active species) is about 10mm. A large variety of primary, secondary and tertiary amines or amides will not replace ammonia. One mol.prop. of adenosine 5′-phosphosulphate reacts with 1 mol.prop. of ammonia to yield 1 mol.prop. each of adenosine 5′-phosphoramidate and sulphate; no AMP is found. The highly purified enzyme does not catalyse any of the known reactions of adenosine 5′-phosphosulphate, including those catalysed by ATP sulphurylase, adenosine 5′-phosphosulphate kinase, adenosine 5′-phosphosulphate sulphotransferase or ADP sulphurylase. Adenosine 5′-phosphoramidate is found in old samples of the ammonium salt of adenosine 5′-phosphosulphate and can be formed non-enzymically if adenosine 5′-phosphosulphate and ammonia are boiled. In the non-enzymic reaction both adenosine 5′-phosphoramidate and AMP are formed. Thus the enzyme forms adenosine 5′-phosphoramidate by selectively speeding up an already favoured reaction.     

Degradation of 5′ adenosine monophosphate in a cell-free system ofEscherichia coli

Sonicated cells ofEscherichia coli contain an enzyme system degrading 5′ adenosine monophosphate (5′ AMP) to hypoxanthine. This enzyme system is located in the fraction sedimenting at 20,000 xg. It has a pH optimum at 8.0. In the fraction sedimenting at 20,000 xg the enzyme activity was inhibited by adenosine triphosphate (ATP). Adenosine and adenine are deaminated by this enzyme preparation to inosine and to hypoxanthine, these activities not being inhibited by ATP.     

Preparation of 5′-deoxy-5′-amino-5′-C-methyl adenosine derivatives and their activity against DOT1L

From a readily available 5-C-Me ribofuranoside, we have realized a reliable route to valuable 5′-deoxy-5′-amino-5′-C-methyl adenosine derivatives at gram scale with confirmed stereochemistry. These adenosine derivatives are useful starting materials for the preparation of 5′-deoxy-5′-amino-5′-C-methyl adenosine derivatives with higher complexity. From one of the new adenosine derivatives, some 5′-deoxy-5′-amino-5′-C-methyl adenosine DOT1L inhibitors were prepared in several steps. Data from DOT1L assay indicated that additional 5′-C-Me group improved the enzyme inhibitory activity.     

Studies on adenosine 3′,5′-monophosphate phosphodiesterase of human erythrocyte membranes

In this work we show the existence of cyclic AMP phosphodiesterase (EC 3.1.4.17) in human erythrocyte membranes and have clarified some properties of the enzyme. In human erythrocytes, about 23% of the total cyclic AMP phosphodiesterase activity is in a membrane-bound form. Although it could be solubilized with Triton X-100 in 5 mM Tris-HCl buffer (pH 8.0), it was not solubilized by a low or high concentration of salt. The enzyme seems to be localized in the cytoplasmic surface, since it is detected in sealed inside-out vesicles of human erythrocyte membranes, but not in intact human erythrocytes. The optimum pH was found to lie between 7.4 and 8.0, and Mg²⁺ was found to be necessary for its activity. Ca²⁺ and calmodulin could not stimulate the activity of this enzyme. Theophylline was a strong inhibitor, but cyclic GMP could not inhibit the enzymic hydrolysis of cyclic [³²P]AMP and this membrane-bound enzyme therefore seems to be specific to cyclic AMP.     

A nucleotide with the properties of adenosine 5′ phosphoramidate from Chlorella cells

We have extracted and purified a nucleotide from cells of $\text{Chlorella}\text{,}\text{pyrenoidose}$ Chick which shares the following properties with adenosine 5′ phosphoramidate; electrophoretic mobility in sodium bicarbonate and in sodium borate buffer (pH 8.0); retention time on high performance liquid chromatography; ultraviolet absorption spectrum at pH 1–2 and 7–9; a yield of one mole each of adenine, ribose, total phosphate and ammonia released at low pH; and formation of adenosine 5′ monophosphate on acidification or treatment with 3′:5′-cyclic-nucleotide phosphodiesterase (EC3.1.4.17). Although formation of APA from its precursor adenosine 5′ phosphosulfate during extraction and purification is not expected this appears to be excluded by the use of low temperature throughout purification and the finding that [¹⁴C] APS added before extraction does not significantly label the adenosine 5′ phosphoramidate isolated. Thus adenosine 5′ phosphoramidate appears to be a normal constituent of $\text{Chlorella}$ cells like the enzyme which forms it: adenylyl sulfate: ammonia adenylyl transferase.     

Dynamic redistribution of paxillin in bovine osteoblasts stimulated with adenosine 5′-triphosphate

Exposure to extracellular 5′-adenosine triphosphate (ATP) is known to induce membrane blebbing. In this study, we investigated the subcellular distribution of the cytoskeletal adaptor protein paxillin in primary bovine osteoblasts upon stimulation with ATP. Cells expressing a fusion protein of green fluorescent protein (GFP) and paxillin were followed by time-lapse video-microscopy after stimulation with 100?μM ATP. Within 100?s, GFP-paxillin became incorporated in numerous de novo formed focal aggregates localized at the cell periphery. The assembly of individual paxillin-containing aggregates occurred with a mean half-life time of <60?s, whereas their disassembly lasted twice as long. Despite the ongoing presence of ATP, the formation of paxillin aggregates was self-limiting within 25?min. Paxillin clustering was preceded by a transient rise in cytoplasmic calcium transients, which peaked already 20?s after adding ATP. The high mobility of paxillin was confirmed by measuring the dissociation rate of GFP-paxillin at mature focal adhesions, demonstrating the presence of a highly mobile fraction with a mean recovery half-life of 8.2?±?1.2?s, followed by a slower phase (53?±?20?s). Thus, both the exchange of paxillin at mature focal adhesions and the increase in intracellular calcium concentrations upon ATP stimulation are very rapid processes, which override the time course of ATP-induced paxillin membrane clustering by one to two orders of magnitude. Our data demonstrate that the transient recruitment of paxillin in membrane protuberances is based on the high intracytoplasmic mobility of unbound paxillin molecules and their rapid focal accumulation.     

Ontogeny of adenosine 3′,5′-monophosphate metabolism in rabbit cerebral cortex

The effects of norepinephrine (NE), histamine (HIST), glutamate, and adenosine, singly and in combinations, on the accumulation of adenosine 3,5-monophosphate (cyclic AMP) in slices of rabbit cerebral cortex were examined using tissue from animals 4 days before to 38 days after birth. A response to NE became visible 2 days before birth and increased to - maximum at 7 days after birth before declining toward the small adult value during the second post-natal week. During this period NE was at least twice as efficacious as isoproterenol, and both - and -adrenergic antagonists had prominent inhibitory effects. Responses to HIST were already apparent 4 days before birth and increased in an irregular fashion thereafter, sometimes exceeding the adult value during the second post-natal week. The response to adenosine was not visible until birth and gradually increased toward the adult value during the entire period examined. Synergistic responses to various combinations of the three agents were first detected at 2 to 4 days before birth. The degree of synergism was larger during the neonatal period than that found in adult tissue; no synergism between HIST and adenosine persisted in the adult. During the first post-natal week, L-glutamate produced very large increases of cyclic AMP accumulation in the presence of either adenosine or histamine plus theophylline; smaller but substantial responses occurred in combination with NE. Responses to glutamate declined progressively after about the tenth post-natal day.     

A general method for visualizing enzymes releasing adenosine or adenosine-5′-monophosphate

Biochemical Genetics -