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     

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.     

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.     

Relationship between cyclic adenosine 3′:5′-monophosphate and germination inCandida albicans

Germination of yeast-like cells ofCandida albicans is preceded by a significant decrease in intracellular levels of cyclic AMP during the early stage of germ-tube induction. These levels increased thereafter as germ-tube formation proceeded. The intracellular concentration of cyclic AMP was measured with a cyclic AMP radioimmunoassay and with a competitive assay method using a cyclic AMP-binding protein. Under inducing conditions, germtube formation was inhibited by the addition of cyclic AMP or compounds that are known to elevate the intracellular cyclic nucleotide concentration.     

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.     

Binding proteins for adenosine 3′:5′-cyclic monophosphate in bovine adrenal cortex

Five peaks of cyclic AMP-binding activity could be resolved by DEAE-cellulose chromatography of bovine adrenal-cortex cytosol. Two of the binding peaks co-chromatographed with the catalytic activities of cyclic AMP-dependent protein kinases (ATP-protein phosphotransferase, EC 2.7.1.37) of type I or type II respectively. A third binding protein was eluted between the two kinases, and appeared to be the free regulatory moiety of protein kinase I. Two of the binding proteins for cyclic AMP, sedimenting at 9S in sucrose gradients, could also bind adenosine. They bound cyclic AMP with an apparent equilibrium dissociation constant (K(d)) of about 0.1mum, and showed an increased binding capacity for cyclic AMP after preincubation in the presence of K(+), Mg(2+) and ATP. The two binding proteins differed in their apparent affinities for adenosine. The isolated regulatory moiety of protein kinase I had a very high affinity for cyclic AMP (K(d)<0.1nm). At low ionic strength or in the presence of MgATP, the high-affinity binding of cyclic AMP to the regulatory subunit of protein kinase I was decreased by the catalytic subunit. At high ionic strength and in the absence of MgATP the high-affinity binding to the regulatory subunit was not affected by the presence of catalytic subunit. Under all experimental conditions tested, dissociation of protein kinase I was accompanied by an increased affinity for cyclic AMP. To gain some insight into the mechanism by which cyclic AMP activates protein kinase, the interaction between basic proteins, salt and the cyclic nucleotide in activating the kinase was studied.     

An effect of insulin on adipose-tissue adenosine 3′:5′-cyclic monophosphate phosphodiesterase

1. 3':5'-Cyclic nucleotide phosphodiesterase activity was measured in homogenates prepared from epididymal fat-pads and isolated fat-cells incubated in the absence and presence of insulin. 2. Homogenates of insulin-treated tissues showed an increase in phosphodiesterase activity compared with controls. No effect of insulin was observed when the hormone was added directly to homogenates. 3. There was kinetic evidence for the presence of two 3':5'-cyclic nucleotide phosphodiesterases in adipose tissue. Insulin raised the maximal velocity of the low-K(m) enzyme and lowered the K(m) of the higher-K(m) enzyme. 4. It is suggested that the effect of insulin on adipose tissue phosphodiesterase accounts for the ability of this hormone to lower cyclic-AMP concentration in the tissue.     

The effect of glucose, insulin and noradrenaline on lipolysis and on the concentrations of adenosine 3′:5′-cyclic monophosphate and adenosine 5′-triphosphate in adipose tissue

1. The deoxyfluoro-d-glucopyranose 6-phosphates were prepared from the corresponding deoxyfluoro-d-glucoses and ATP by using hexokinase. 2. 3-Deoxy-3-fluoro- and 4-deoxy-4-fluoro-d-glucose 6-phosphate were substrates for glucose phosphate isomerase, and in addition the products of this reaction, 3-deoxy-3-fluoro- and 4-deoxy-4-fluoro-d-fructose 6-phosphate respectively, were good substrates for phosphofructokinase. 3. Some C-2-substituted derivatives of d-glucose 6-phosphate were found to be competitive inhibitors of glucose phosphate isomerase. 4. The possible role of the hydroxyl groups in the binding of d-glucose 6-phopshate to glucose phosphate isomerase is discussed.     

Effect of guanosine 3′:5′-monophosphate on the hydroosmotic activity of adenosine 3′:5′-monophosphate in toad urinary bladder

Guanosine 3′:5′-monophosphate has a slight hydroosmotic effect on toad urinary bladder. Furthermore, this nucleotide strongly inhibits the responses to 3′:5′-adenosine monophosphate and oxytocin. The response to an increase in medium tonicity is not modified by the guanosine nucleotide. A role for guanosine 3′:5′-monophosphate in the regulation of water permeability in toad urinary bladder is proposed.     

Stimulation of calcium efflux from rat liver mitochondria by adenosine 3′5′ cyclic monophosphate

Summary The uptake or release of Ca²⁺ from rat liver mitochondria was studied by means of a sensitive Ca-electrode. It was found that using palmitoyl coenzyme A together with carnitine and ATP as substrates that Ca²⁺ was released gradually from mitochondria by adenosine 35 cyclic monophosphate. The effect was obtained with either mitochondria preloaded with Ca²⁺ or with their physiological content of Ca²⁺. No such release was obtained with the usual substrates used to provide energy for Ca²⁺ uptake by mitochondria.     

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.     

Involvement of adenosine 3′:5′-cyclic monophosphate in lipid mobilization in Locusta migratoria

The rôle of cyclic AMP in hormone-induced lipid mobilization in Locusta migratoria was investigated. Injection of a corpus cardiacum extract into adult female locusts resulted in an increased level of cyclic AMP in the fat body. The cAMP concentration is maximal at about 5 min of incubation and returns to the resting level after about 10 min. The dose-response curve is linear up to about 0.01 corpus cardiacum pair equivalents.Dibutyryl-cyclic AMP mimics the lipid mobilizing effect of corpus cardiacum extract. After flight the cyclic AMP concentration in fat body increased. Injection of corpus cardiacum extract had no effect on flight muscle cyclic AMP concentration.     

Anti-anabolic effects of adenosine 3′:5′-cyclic monophosphate. Inhibition of protein synthesis

1. Evidence is presented that cyclic AMP inhibits the incorporation of l-[4,5-(3)H]leucine into protein in a cell-free system from rat liver. This inhibition occurs after aminoacyl-tRNA formation. 2. Microsomal fractions, isolated after the incubation of postmitochondrial supernatant with cyclic AMP and ATP, show a diminished ability to synthesize protein. Both cyclic AMP and ATP are required for this effect. 3. A possible physiological role for the anti-anabolic action of cyclic AMP is discussed in terms of the control of gluconeogenesis.