Responses of the rectum and oesophagus of the snail Helix aspersa to purine nucleotides and nucleosides

1. Purine compounds were examined for pharmacological activity in the rectum and oesophagus of the garden snail Helix aspersa.2. In the rectum, adenosine, AMP, ADP and ATP (above 10μM) and acetylcholine (above 1 nM) consistently caused concentration-dependent contractions. The slope of the dose-response curve for ADP in the rectum was significantly steeper than for the other purine compounds. The contractile responses to the nucleotides and acetylcholine, but not adenosine, were selectively potentiated by physostigmine (1μM). Atropine (1 μM) and tubocurarine (30 μM) failed to block the responses to the purines or acetylcholine.3. In the oesophagus, adenosine, AMP, ADP and ATP (above 10 μM) and acetylcholine (above 1 nM) caused concentration-dependent contractions that were antagonised by atropine (l μM). Tubocurarine (30 μM) failed to block the responses to the purine compounds or acetylcholine. Physostigmine (1 μM) potentiated the responses to ADP and acetylcholine but not ATP, AMP or adenosine.4. In both the rectum and the oesophagus, the synthetic analogues of purine compounds inclucling 2-chloroadenosine, α, β -methylene ATP and 2-methylthio ATP were inactive up to a concentration of 100 μM.5. Electrical field stimulation of the rectum and oesophagus produced consistent contractions which were unaffected by atropine (1 μM), tubocurarine (30 μM) or physostigmine (1 μM). These responses were not modulated by any of the purine compounds or their stable analogues.6. The responses obtained appear novel even within known invertebrate purinergic systems, suggesting a differentiation of purinoceptor subtypes in this species. There is evidence in the rectum for AMP, ADP and ATP causing the release of acetylcholine; physostigmine potentiated responses to AMP, ADP and ATP, but not to adenosine. This indicates that activity may be mediated via different types of purinoceptors, perhaps equivalent to the P₁- and P₂-purinoceptors identified in vertebrates.     

The interaction of aflatoxins with purines and purine nucleosides

From measurements of thermal hyperchromicity and the behaviour of an aflatoxin-DNA mixture on a Sephadex column it was concluded that aflatoxin B(1) is capable of weak binding to single-stranded DNA. The interactions of the aflatoxins (B(1), G(1) and G(2)) with nucleosides result in difference spectra and suggest that the purine bases and the amino group play a role in the binding of all the aflatoxins to DNA.     

Urinary excretion of purines,purine nucleosides,and pseudouridine in immunodeficient children

Studies have been carried out using ion-exchange analysis for determination of urinary purines, pyrimidines, and nucleosides in children with immunodeficiency disorders. Using cation and anion-exchange techniques, the following compounds of urine have been quantitatively determined: deoxyadenosine, adenosine, adenine, pseudouridine, 7-methylguanine, N²,N²-dimethylguanosine, hypoxanthine, and xanthine. Excretion levels of these compounds did not differ significantly from normal values in six children with various immunodeficiency diseases, excluding severe combined immunodeficiency. However, of the seven children studied with severe combined immunodeficiency disease (normal adenosine deaminase), four showed increased excretion levels for one or more of the compounds studied. A germ-free child with severe combined immunodeficiency had lower excretion levels than the mean normal value for most of these same compounds. The possibility is considered that the increased excretion levels noted may be a consequence of repeated episodes of infection in most children with severe combined immunodeficiency.     

Fluoride ion catalyzed alkylation of purines, pyrimidines, nucleosides and nucleotides using alky halides.

Alkyl halides react rapidly with purines and pyrimidines in the presence of fluoride ion. Alkylation of thymidine leads to novel dimeric nucleoside derivatives bridged through N3. Alkylation of thymidine mono and dinucleotides leads to alkylation at the base (N3) as well as diester and triester formation at the phosphate.     

Ribose modified nucleosides and nucleotides as ligands for purine receptors

Molecular modeling of receptors for adenosine and nucleotide (P2) receptors with docked ligand, based on mutagenesis, was carried out. Adenosine 3',5'-bisphosphate derivatives act as selective P2Y1 antagonists/partial agonists. The ribose moiety was replaced with carbocyclics, smaller and larger rings, conformationally constrained rings, and acyclics, producing compounds that retained receptor affinity. Conformational constraints were built into the ribose rings of nucleoside and nucleotide ligands using the methanocarba approach, i.e. fused cyclopropane and cyclopentane rings in place of ribose, suggesting a preference for the Northern (N) conformation among ligands for P2Y1 and A1 and A3ARs.     

Uptake and metabolism of purine nucleosides and nucleotides in isolated frog skeletal muscle.

When isolated frog skeletal muscles were incubated with ¹⁴C-labeled adenosine, the nucleoside was rapidly taken up by the cells and was either immediately incorporated into adenine nucleotides or deaminated to inosine. Incorporation was predominant at low (micromolar) concentrations whereas, deamination was the major route of metabolism at high (millimolar) concentrations. When muscles were incubated with ¹⁴C-labeled inosine the nucleoside, after entry into the cells, was metabolized to a lesser extent than adenosine. ATP and hypoxanthine were the major products of its metabolism. Intracellular concentrations were calculated using ³H-labeled sorbitol to measure the extracellular space.Because of its lower rate of intracellular metabolism inosine was used to investigate the characteristics of the nucleoside transport system. The uptake of inosine was saturable at high concentrations and was specifically inhibited by the presence of adenosine or uridine in the incubation media. Persantin, a well known specific inhibitor of nucleoside transport, also competitively inhibited inosine uptake, as did theophylline [1, Woo et al. Can J. Physiol. Pharmacol. $\text{52}$, 1063, 1974]. These data, along with the knowledge that in a well-oxygenated muscle, inosine entry follows a downhill chemical potential gradient, strongly support the view that the transport mechanism is facilitated diffusion.The muscle cell membrane does not appear to be permeable to ¹⁴C-labeled ATP under the conditions studied. Investigations of the permeability to the major extracellular degradation products of ATP suggest that AMP was the compound most likely to cross the cell membrane.     

Interconversion and uptake of nucleotides, nucleosides, and purine bases by the marine bacterium MB22.

Whole cells and isolated membranes of the marine bacterium MB22 converted nucleotides present in the external medium rapidly into nucleosides and then into bases. Nucleosides and purine bases formed were taken up by distinct transport systems. We found a high-affinity common transport system for adenine, guanine, and hypoxanthine, with a Km of 40 nM. This system was inhibited noncompetitively by purine nucleosides. In addition, two transport systems for nucleosides were present: one for guanosine with a Km of 0.8 microM and another one for inosine and adenosine with a Km of 1.4 microM. The nucleoside transport systems exhibited both mixed and noncompetitive inhibition by different nucleosides other than those translocated; purine and pyrimidine bases had no effect. The transport of nucleosides and purine bases was inhibited by dinitrophenol or azide, thus suggesting that transport is energy dependent. Inside the cell all of the substrates were converted mainly into guanosine, xanthine, and uric acid, but also anabolic products, such as nucleotides and nucleic acids, could be found.     

Preservation of red blood cells with purines and nucleosides. II. Uptake and utilization of purines and nucleosides by stored red blood cells

The uptake of adenine, guanine, guanosine and inosine by stored red cells was investigated in whole blood and red cell resuspensions at initial concentrations of 0.25, 0.5 and 0.75 mM for adenine and 0.5 mM for the other additives using a rapid ion-exchange chromatographic microanalysis of purines and nucleosides in plasma and whole blood. Increasing adenine concentrations from 0.25 to 0.75 mM in blood elevated the adenine uptake from 0.3 up to 0.8 mmol/l red cells during 2 hours after collecting blood. The intra-/extracellular distribution ratio changed from 1 : 1.3 to 1: 1.7. Some 2 hours after withdrawing blood into CPD--solution with purines and nucleosides the uptake of adenine and guanine resulted in 40 per cent and 70 per cent respectively and of guanosine and inosine in 80 and 90 per cent respectively. The replacement of plasma by a resuspending solution gave the same uptake rates for purines and nucleosides. The nucleosides were rapidly split to purines and R-1-P and disappeared from blood during one week. Adenine and guanine were utilized to 80 to 90 per cent only after 3 weeks. During the same period the utilization of guanine was smaller by 40 per cent than that of adenine due to the different activity of the purine nucleoside phosphorylase for these substrates. The plasma of all analyzed blood samples contained hypoxanthine and inosine, but guanine and guanosine were detected only in those samples to which one of them was added. After 3 weeks of storage the highest concentration of hypoxanthine was found in CPD-AI blood with 600 microM in plasma and the highest concentration of synthesized inosine in CPD-AG blood with a concentration of 100 microM in plasma. Three ways of utilization of purines by stored red cells were discussed : the synthesis of nucleotide monophosphates, the formation of nucleosides, and the deamination. The portions of these ways change during storage. The most effective concentrations of adenine and guanosine in stored blood seems to be 0.25 and 0.5 mM respectively. The full utilization of the nucleoside requires the addition of inorganic phosphate.     

Preservation of red blood cells with purines and nucleosides. III. Synthesis of adenine, guanine, and hypoxanthine nucleotides

Purine nucleotides of fresh human red cells and of red cells during storage at 4 degrees and 25 degrees C with additions of adenine, guanine, guanosine and inosine were estimated by HPLC. Six nucleotides were found in red cells: ATP, ADP, AMP, GTP, GDP, and IMP. The adenine nucleotides represented 92 per cent of the total purine nucleotides, guanine nucleotides 7 per cent and IMP less than 1 per cent. In red cells stored with adenine the total concentration of purine nucleotides increased to 125 per cent of the normal value. An adenine-free but guanine and guanine + inosine containing medium caused a decrease of the concentration of purine nucleotides by 10 to 20 per cent. When red cells were stored without adding guanine or guanosine the content of the guanine nucleotides decreased from 0.32 to 0.17 mumol/g Hb due to the decrease in the GTP content, but the GDP concentration increased slightly. In CPD-AG blood, however, the concentration of guanine nucleotides increased considerably up to 0.6 mumol/g Hb. IMP was estimated in all investigated stored red cells. In CPD-A and in CPD-AG blood 0.4 mumol/g Hb were produced during 3 weeks of storage, but twice of that in CPD-AI blood. The principles of the synthesis and the degradation of purine nucleotides in stored red cells are discussed in detail.     

Preservation of resuspended red cell concentrate. Analysis of purines, nucleosides and nucleotides in stored red cells

Purine nucleotides of red blood cells (RBC) during storage in two different media with addition of adenine/nicotinamide (NAP) or adenine/guanosine (CDS-AG) were estimated by HPLC. Synthesis of guanine nucleotides reached a maximum after 14 days in RBC stored with adenine/guanosine. The higher adenine concentration in the NAP solution (3 mmol/l) did not increase adenine consumption and the ATP-level of the erythrocytes. The adenylate energy charge (AEC) of RBC decreased from 0.91 to 0.63 during 42 days of storage in CDS-AG solution.     

Extraction and measurement of myocardial nucleotides, nucleosides, and purine bases by high-performance liquid chromatography

Nucleotides, nucleosides, and purine bases were extracted from human endomyocardial biopsies, freeze-clamped rat hearts, and porcine coronary sinus plasma. Perchloric acid extracts were neutralized with Freon-trioctylamine and analyzed at 250 nm by reverse-phase ion-pairing high-performance liquid chromatography. To achieve the sensitivity necessary for analyzing small (1-3 mg wet wt) tissue samples, a small-bore, 2.1-mm-internal-diameter, C18, 5-micron reverse-phase column and a flow rate of 0.2 ml/min were used. All of the myocardial nucleotides and AMP degradation products were resolved in a total separation time of 27 min with 30 mM KH2PO4, 7.5 mM tetrabutylammonium phosphate buffers, and binary pH and acetonitrile gradients.     

Interaction between NiCl2, and nucleobases, nucleosides and nucleotides

The interaction between NiCl, and nucleobases, nucleosides and nucleotides has been studied by UV-Vis difference spectrophotometry, graphite furnace atomic absorption spectrophotometry, IR spectroscopy and high pressure liquid chromatography using the technique of continuous variation. The proposed structures of the complexes formed were optimised and their electronic and vibrational spectra generated using the molecular modelling program HyperChem 5. Ni2+ reacts with guanine, 2'-dGMP, GMP, adenine and AMP to form 1:1 complexes Ni(Guanine)(H2O)5, Ni(2'-dGMP)(H2O)5, Ni(GMP)(H2O)5, Ni(Adenine)(H2O)5, and Ni(AMP)(H2O)5 respectively. In these complexes, Ni2+ is believed to be bonded to the N7 atom of adenine and guanine.     

Uric acid synthesis by rat liver supernatants from purine bases,nucleosides and nucleotides. Effect of allopurinol

The synthesis of uric acid from purine bases, nucleosides and nucleotides has been measured in reaction mixtures containing rat liver supernatant and each one of the following compounds at 1 mM concentration (except xanthine, 0·5 mM and guanosine and guanine, 0·1 mM). The rates of the reaction, expressed as nanomoles of uric acid synthesized g^?1 of wet liver min^?1 were: ATP, 10; ADP, 37; AMP, 62; adenosine, 108; adenine 6; adenylo-succinate, 9; IMP 32; inosine, 112; hypoxanthine, 50; GTP, 19; GDP, 19; GMP, 27; guanosine, 34; guanine, 72; XMP, 10; xanthosine, 24; xanthine, 144. These figures divided by 55 correspond to nanomoles of uric acid synthesized min^?1 per mg^?1 of protein. The rate of synthesis of uric acid obtained with each one of those compounds at 0·1 and 0·05 mM concentrations was also determined. ATP (1 nM) strongly inhibited uric acid synthesis from 0·05 mM AMP (91 per cent) and from 0·05 mM ADP (88 per cent), but not from adenosine. CTP or UTP (1 mM ) also inhibited (by more than 90 per cent) the synthesis of uric acid from 0·05 mM AMP. Xanthine oxidase was inhibited by concentrations of hypoxanthine higher than 0·012 mM. The results favour the view that the level of uric acid in plasma may be an index of the energetic state of the organism. Allopurinol, besides inhibiting uric acid synthesis, reduced the rate of degradation of AMP. The ability of crude extracts to catabolize purine nucleotides to uric acid is an important factor to be considered when some enzymes related to purine nucleotide metabolism, particularly CTP synthase, are measured in crude liver extracts.     

A new synthesis of 5''-deoxy-8,5''-cyclo-adenosine and -inosine: conformationally-fixed purine nucleosides (nucleosides and nucleotides. XVI).

A versatile method for the synthesis of 5'-deoxy-8,5'-cycloadenosine, a conformationally-fixed "anti" type of adenosine, was presented. Irradiation of 2', 3'-O-isopropylidene-5'-deoxy-5'-phenylthioadenosine with 60W Hg vapor lamp afforded 2',3'-O-isopropylidene-5'-deoxy-8,5'-cycloadenosine in high yield. The use of other 5'-alkylthio derivatives also gave the cycloadenosine, though the yields were rather poor. Deacetonation of the cyclocompound with 0.1N HCl gave 5'-deoxy-8,5'-cycloadenosine. The cycloinosine derivative was similarly prepared. The nmr, mass and CD spectra of 5'-deoxy-8,5'-cycloadenosine were given and discussed with the previously reported results.