The Na+/H+ Exchanger Present in Trout Erythrocyte Membranes is Not Responsible for the Amiloride-insensitive Na+/Li+ Exchange Activity

The protein responsible for the Na⁺/Li⁺ exchange activity across the erythrocyte membrane has not been cloned or isolated. It has been suggested that a Na⁺/H⁺ exchanger could be responsible for the Na⁺/Li⁺ exchange activity across the erythrocyte membrane. Previously, we reported that in the trout erythrocyte, the Li⁺/H⁺ exchange activity (mediated by the Na⁺/H⁺ exchanger βNHE) and the Na⁺/Li⁺ exchange activity respond differently to cAMP, DMA (dimethyl-amiloride) and O₂. We concluded that the DMA insensitive Na⁺/Li⁺ exchange activity originates from a different protein. To further examine these findings, we measured Li⁺ efflux in fibroblasts expressing the βNHE as the only Na⁺/H⁺ exchanger. Moreover, the internal pH of these cells was monitored with a fluorescent probe. Our findings indicate that acidification of fibroblasts expressing the Na⁺/H⁺ exchanger βNHE, induces a Na⁺ stimulated Li⁺ efflux activity in trout erythrocytes. This exchange activity, however, is DMA sensitive and therefore differs from the DMA insensitive Na⁺/Li⁺ exchange activity. In these fibroblasts no significant DMA insensitive Na⁺/Li⁺ exchange activity was found. These results support the hypothesis that the trout erythrocyte Na⁺/Li⁺ exchange activity is not mediated by the Na⁺/H⁺ exchanger (βNHE) present in these membranes. Received: 6 December 1996/Revised: 11 August 1997     

An adenosine 3':5'-monophosphate-adenosine binding protein from mouse liver. Factors affecting the activation of the binding protein by adenosine 5'-triphosphate.

A cyclic AMP-adenosine binding protein, whose binding sites are activated by preincubation in the presence of Mg⁺-ATP, has been purified to apparent homogeneity from mouse liver (P.M. Ueland and S.O. Døskeland, 1977, J. Biol. Chem.,252, 677–686). The degree of activation of both the cyclic AMP binding site and a high-affinity site for adenosine depends on the concentration of ATP during the preincubation. The velocity and the degree of activation are dependent on the temperature and the presence of Mg²⁺ and K⁺. The NH₄⁺ ion can be substituted for K⁺, whereas Na⁺ is inefficient. Low pH promotes the conversion from the inactive to the active form. The apparent affinity for adenosine to the high-affinity site for this adenine derivative and the affinity for cyclic AMP to the site specific for this nucleotide are independent of the degree of activation as judged from the slope of Scatchard plots. The activation of the cyclic AMP binding site by ATP (6 mm) was determined at pH 7 in the presence of 10 μm cyclic AMP, AMP, ADP, or adenosine. Adenosine specifically inhibits the activation and does not promote the inactivation of the binding protein. The possibility that the apparent inhibition of activation was effected by interference with cyclic AMP binding by adenosine was ruled out.     

Fluorescence spectra,fluorescence quantum yield and dissociation constant of sarafloxacin

In this study, the fluorescence spectra of sarafloxacin (SAR) under different pH conditions were investigated to determine the structural changes due to protonation that result from change in pH. At pH < 1.02, SAR exists in the H₃L²⁺ form for which the maximum fluorescence emission wavelength was about 455 nm. At pH 1.87–4.94, SAR exists in the H₂L⁺ form in which H₃L²⁺ loses one proton in the nitrogen molecule at the 1‐position in the quinoline ring. Fluorescence intensity was strong and steady and the maximum emission wavelength was 458 nm. At pH 7.14–9.30, the maximum emission wavelengths were gradually blue shifted to 430 nm with increase in pH, here SAR exists in the form of a bipolar ion HL in which H₂L⁺ loses a carboxyl group proton. At pH > 11.6, HL transforms into anionic L^? in which HL loses one proton from the piperazine ring, leading to a decrease in fluorescence intensity, and the maximum emission wavelength was red shifted to approximately 466 nm. The two‐step dissociation constant pKa for SAR was calculated, pK _a1 was 6.06 ± 0.37 and pK _a2 for SAR was 10.53 ± 0.19. In a pH 3.62 buffer solution with quinine sulfate as the reference, the fluorescence quantum yield of SAR at the maximum excitation wavelength of 276 nm was 0.09.     

Enhanced release of adenosine under cell-damaging conditions in the developing and adult mouse hippocampus

The inhibitory neuromodulator adenosine has been thought to act as an endogenous neuroprotectant against cerebral ischemia and neuronal damage. The release of preloaded [³H]adenosine from hippocampal slices from developing (7-day-old) and adult (3-month-old) mice was characterized using a superfusion system under various cell-damaging conditions, including hypoxia, hypoglycemia, ischemia, oxidative stress, and the presence of free radicals and metabolic poisons. The release of adenosine was greatly potentiated under the above conditions at both ages, with free radicals, metabolic poisons, and ischemia generally having the strongest stimulatory effects. Depolarization by K⁺ ions (50 mM) could then evoke more release of adenosine only in the immature hippocampus. Omission of Ca²⁺ from the superfusion media had no effect on the ischemia-induced release in the adults, indicating that it occurs by a Ca²⁺-independent system. In contrast, the release in the immature hippocampus was partially dependent on extracellular Ca²⁺. Furthermore, the ischemia-induced adenosine release was reduced in Na⁺-deficient media and enhanced by ouabain at both ages, pointing to the involvement of Na⁺-dependent transporters. The release was also reduced by Cl^– channel blockers, thus indicating that a part of the evoked release occurs through anion channels. Another inhibitory neuromodulator and cell volume regulator, taurine, was seen to enhance adenosine release in ischemia at both ages. The simultaneous release of taurine and adenosine under cell-damaging conditions could constitute an important protective mechanism against excessive amounts of excitatory amino acids, counteracting their harmful effects and preventing excitation from reaching neurotoxic levels.     

Influence of temperature acclimatization on the ionic activation of goldfish intestinal adenosine triphosphatase

1. An adenosine triphosphatase membrane system, dependent on Mg²⁺ and activated further by Na⁺+K⁺, was prepared from goldfish anterior intestine by differential centrifugation of homogenized intestinal scrapings. 2. The affinity of this preparation for Na⁺ in the presence of K⁺+Mg²⁺, for K⁺ in the presence of Na⁺+Mg²⁺ and for Mg²⁺ alone, measured at 37°, did not depend on the previous environmental temperature of the fish. When Na⁺+K⁺ were added to preparations from 8°-acclimatized fish the affinity for Mg²⁺ increased; this was not seen with preparations from 30°-acclimatized fish. 3. Part of the Mg²⁺-activated adenosine triphosphatase was inhibited by Na⁺ and the amount of inhibition appeared to increase at high acclimatization temperatures. 4. This Na⁺-inhibited adenosine triphosphatase was separated from the (Na⁺+K⁺)-activated enzyme by centrifugation on sucrose density gradients. 5. Preparations from 8°-acclimatized fish contained less Mg²⁺-activated and more (Na⁺+K⁺)-activated adenosine triphosphatase than did similar fractions from 30°-acclimatized fish. 6. Acclimatization to different environmental temperatures might involve one form of adenosine triphosphatase changing to another. The origin of various membranes seen in microsomal fractions must, however, be established before this hypothesis can be tested further.     

Radioimmunoassay for N6(methylnitroso)adenosine: production and characterization of antibodies

Antibodies specific for N⁶(methylnitroso)adenosine have been produced in rabbits and a sensitive radioimmunoassay was developed. The nitroso group is immunodominant; 50% inhibition of the binding of [³H]N⁶(methylnitroso)adenosine to antibody was obtained with 9.6 pmoles of N⁶(methylnitroso)adenosine and 200 nmoles of N⁶-methyladenosine. Adenosine was essentially inactive. After nitrosation, N⁶(methylnitroso)adenosine can be detected only in those RNA molecules known to contain N⁶-methyladenosine.     

N6-methyl adenosine in siRNA evades immune response without reducing RNAi activity

Abstract

siRNA is a powerful method to suppress specific gene expression and has recently been utilized for molecular biology as well as medicine. However, introduction of dsRNA stimulates immune-responses as side-effects. In the present study, we utilized N⁶-methyl adenosine, one of the natural modified nucleosides, instead of adenosine in siRNA. When adenosine in the passenger or guide strand of siRNA was completely replaced with N⁶-methyl adenosine, the immune response against siRNA was evaded without any reduction in RNAi activity. This knowledge will promote the medical application of siRNA and enhance our understanding on cellular discrimination of non-self and self dsRNA.     

Location of adenosine release and adenosine A2 receptors to rat striatal neurons

Three types of striatal lesions were performed to determine the site of adenosine synthesis and release and the location of adenosine A2 receptors: decortication; injection of 6-hydroxydopamine (6-OHDA) into the median forebrain bundle; and injection of kainic acid into the striatum. The parameters measured in the striatum were content of adenosine, activation of adenylate cyclase by N⁶-(L-phenylisopropyl) adenosine (PIA) and release of endogenous adenosine from a perfused slice. Decortication and 6-OHDA had only minimal affects on the parameters measured. In contrast, kainic acid injection into the striatum decreased the content of adenosine, the release of adenosine from a slice preparation and diminished the ability of PIA to activate adenylate cyclase. We postulate that neurons which synthesize and release adenosine, originate in the striatum. The adenosine receptors appear to be of the adenosine A2 type and they may be located on adjacent neurons or on the adenosine releasing neurons themselves.     

Phytohormone effects on hydrolytic activity of phosphohydrolases in red beet (Beta vulgaris L.) tonoplasts

The effects of indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellic acid (GA₃) and kinetin on the hydrolytic activity of proton pumps (adenosine triphosphatase, H⁺-ATPase, pyrophosphatase, H⁺-PPase) of tonoplasts isolated from stored red beet (Beta vulgaris L. cv. Bordo) roots were studied. Results suggest that the phytohormones can regulate the hydrolytic activities of H⁺-ATPase and H⁺-PPase of the vacuolar membrane. Each of the proton pumps of the tonoplast has its own regulators in spite of similar localization and functions. IAA and kinetin seem to be regulators of the hydrolytic activity for H⁺-PPase whereas for H⁺-ATPase it may be GA₃. Stimulation of enzyme activity by all hormones occurred at concentrations of 10^–6 to 10^–7 M.Abbreviations IAA indole-3-acetic acid - ABA abscisic acid - GA₃ gibberellic acid - H⁺-ATPase adenosine triphosphatase - H⁺-PPase pyrophosphatase - ATP adenosine triphosphate - Tris Tris (hydroxymethyl)-aminomethane - MES (2[N-Morpholino]) ethane sulfonic acid - EDTA ethylene diamine tetraacetic acid - P_i inorganic phosphate     

A study of the interactions among adenosine triphosphate, epinephrine, and magnesium ions

The interactions among adenosine triphosphate, Mg⁺², and epinephrine at pH's below 7.0 have been studied by observing the effects of these interactions on the chemical shifts and line widths of their ¹H and ³¹P nuclear magnetic resonance spectra. Mg⁺² is tightly bound by the β- and γ-phosphate groups of adenosine triphosphate and there is a weak association between this chelate and epinephrine. In the ternary complex, the aromatic ring of epinephrine overlaps the purine ring of adenosine triphosphate and there appears to be an ionic interaction between the protonated amino group and the α-phosphate of adenosine triphosphate. It was also found that dichloroisoproterenol forms essentially the same type of ternary complex.     

Na+ / adenosine co-transport in Vibrio parahaemolyticus

Adenosine transport in Vibrio parahaemolyticus was studied. Na⁺ greatly stimulated adenosine uptake. Addition of adenosine to a cell suspension under anaerobic conditions elicited Na⁺ uptake, and the Na⁺ uptake was inhibited by monensin, an Na⁺ ionophore. Imposition of an electrochemical potential of Na⁺ or a membrane potential in energy-depleted cells elicited adenosine uptake. Therefore, adenosine transport in this organism was concluded to proceed by an Na⁺ / adenosine co-transport mechanism. The Na⁺ / adenosine co-transport system was induced when cells were grown in the presence of adenosine, and repressed by glucose. Although Na⁺ uptake elicited by adenosine was reduced by glucose, it was enhanced by methyl α-glucoside, which reduced the intracellular ATP level. Thus, the effects of glucose and the glucoside on the Na⁺ / adenosine co-transport system did not seem to be due to inducer exclusion, but to be related to the intracellular ATP level.     

Protonation and quaternization of 1, N6-ethenoadenosine: What are the species responsible for the fluorescence of 1, N6-ethenoadenosine?

Methylation of 1,N⁶-ethenoadenosine (εAdo) gives a mixture of N1- and N9-quaternized methyl-3-β-D -ribofuranosylimidazo[2,1-i] purinium salts (m¹εAdo⁺ and m⁹εAdo⁺, respectively). The ratio of the two forms of the protonated εAdo [H¹εAdo⁺]/[H⁹εAdo⁺] has been estimated to be approximately 0.10 by comparing the uv absorption spectra of the protonated species of εAdo and the two nontautomerizable model compounds. In relation to a study on the protonation effect on the fluorescence of εAdo, we have now determined the effect of quaternization on the fluorescence spectra at 293 and 77 K. We have found that m¹εAdo⁺ and m⁹εAdo⁺ are both fluorescent, and the high degree of coincidence between the fluorescence spectra of εAdo and m¹εAdo⁺ at pH 7 is noted. The m¹εAdo⁺ singlet form is a more efficient fluorescer than the m⁹εAdo⁺ ion at room temperature (quantum yields of 0.43 and 0.11, respectively). All the results which are presented in this paper are consistent with the picture that there exist more than one species responsible for the fluorescence of εAdo, depending on the environment of the molecule in aqueous solution (temperature and pH of solvent).     

Luminescence analysis of SrGa2Si2O8: RE3+ (RE = Dy,Tm) phosphors

This article reports on the luminescence properties of rare earth (Dy³⁺ and Tm³⁺)ions doped SrGa₂Si₂O₈ phosphor were studied. SrGa₂Si₂O₈phosphors weresynthesizedby employing solid state reaction method.From the measured X‐ray diffraction (XRD) pattern of the samplemonoclinic phase structure has been observed. Thermoluminescenceand Mechanoluminescence properties of the γ‐ray irradiated samples have been studied. Photoluminescence spectra of Dy³⁺ activated SrGa₂Si₂O₈phosphor has been measured with an excitation wavelength at 348 nm,and it shows two emission bands at 483 and 574 nm due to ⁴F_9/2 → ⁶H_15/2 and ⁴F_9/2 → ⁶H_13/2 transitions respectively. Whereas the photoluminescence spectra of Tm³⁺ activated SrGa₂Si₂O₈ phosphor has been measured with an excitation wavelength at 359 nm and it exhibits two emission bands at 454 and 472 nm due to ¹D₂ → ³F₄ and¹G₄ → ³H₆ transitions respectively. In thermoluminescence study, γ‐irradiatedthermoluminescence glow curve of SrGa₂Si₂O₈:Dy³⁺ phosphor shows two well defined peaks at 293 °C (peak1)and 170 °C (peak2) whereas thermoluminescence glow curve of SrGa₂Si₂O₈:Tm³⁺ phosphor shows peaks at 292 °C (peak1) and 184 °C (peak2) indicating that two sets of traps are being activated within the particular temperature range and the trapping parameters associated with the prominent glow peaks of SrGa₂Si₂O₈:Dy³⁺ and SrGa₂Si₂O₈:Tm³⁺ are calculated using Chen's peak shape and initial rise method.From the Mechanoluminescence study, only one glow peak has been observed. Copyright © 2016 John Wiley & Sons, Ltd.     

Active extrusion of protons into deionized water by roots of intact maize plants

The investigations were focussed on the question as to whether roots of intact maize plants (Zea mays L. cv Blizzard) release protons into deionized H₂O. Plants in the six to seven leaf stage depressed the pH of deionized H₂O from 6 to about 4.8 during an experimental period of 4 hours. Only one-third of the protons released could be ascribed to the solvation of CO₂ in H₂O. The main counter anions released were Cl⁻, NO₃⁻, and SO₄²⁻. At low temperature (2°C), the H⁺ release was virtually blocked while a relatively high amount of K⁺ was released. The presence of K⁺, Na⁺, Ca²⁺, and Mg²⁺ in the external solution increased the H⁺ secretion significantly. Addition of vanadate to the outer medium inhibited the H⁺ release while fusicoccin had a stimulating effect. Substituting the nutrient solution of deionized H₂O resulted in a substantial increase of the membrane potential difference from −120 to −190 millivolts. The experimental results support the conclusion that the H⁺ release by roots of intact maize plants is an active process driven by a plasmalemmalocated ATPase. Since the net H⁺ release was not associated with a net uptake of K⁺, it is unlikely to originate from a K⁺/H⁺ antiport.     

Effect ofin vivo andin vitro application of the cytokinin N6 -(m-hydroxybenzyl)adenosine on respiration and membrane transport processes in sugar beet

Sugar beet grown in pots was sprayed with N⁶-(m-hydroxybenzyl)adenosine, (mOH)- [9R]BAP, one of the synthetic cytokinins. Root tissue was then examined for respiration and for H⁺-adenosinetriphosphatase activity and both leaf and root tissue served as the object for 6-deoxy-D-glucose and 2-aminoisobutyric acid uptake estimations. Treatment with (mOH)[9R]BAP depressed the uptake of oxygen by the roots of both young and old plants by 17 – 30 % while addition of (mOH)[9R]BAP to the respiring slices decreased it by 10 – 23 %. Uptake of 6-deoxy-D-glucose was mostly diminished byin vivo spraying with the cytokinin (by up to 12 % in leaves and by up to 60 % in roots), as well as by adding it to the experimental vessel (insignificantly in the leaves but by up to 80 % in the roots). The H⁺-ATPase activity was stimulated bothin vivo andin vitro appreciably in young plants but not at all in plants at the end of their vegetation period. Acknowledgement: The work described here was supported by grant No. 501/94/0413 of the Grant Agency of the Czech Republic     

Inhibition of adenosine triphosphatase, 5-hydroxytryptamine transport and proton-translocation activities of resealed chromaffin-granule `ghosts''

1. Highly purified resealed chromaffin-granule `ghosts' were assayed for ATPase and ATP-driven H<sup>+</sup>-translocation and 5-hydroxytryptamine-uptake activities, and for 5-hydroxytryptamine uptake driven by an imposed transmembrane H<sup>+</sup>-gradient. The effects of several inhibitors on these activities were studied. 2. Dicyclohexylcarbodi-imide inhibits all of these activities, but not in parallel; at low concentrations it decreases the permeability of the membrane to protons. 3. 4-Chloro-7-nitrobenzofuran (Nbf-Cl) and silicotungstate inhibit ATP-dependent activities, without effect on 5-hydroxytryptamine uptake driven by an imposed H<sup>+</sup>-gradient. 4. Tributyltin chloride inhibits all of the activities. 5. Treatment of the `ghosts' with low concentrations of urea inhibits 5-hydroxytryptamine uptake and ATP-dependent generation of a transmembrane H⁺-gradient, without inhibiting ATPase activity. 6. Nbf-Cl and silicotungstate are without effect on the rate of leakage of 5-hydroxytryptamine from preloaded `ghosts', whereas dicyclohexylcarbodi-imide and tributyltin chloride accelerate the rate of leakage. 7. Treatment of the membranes with ¹⁴C-labelled Nbf-Cl labels several proteins; membranes treated with dicyclohexyl[¹⁴C]carbodi-imide are labelled predominantly in a protein of low molecular weight, which may be analogous to the mitochondrial H⁺-conducting proteolipid. 8. It is concluded that Nbf-Cl and silicotungstate inhibit the H⁺-translocating ATPase of the granule membrane; that dicyclohexylcarbodi-imide inhibits the ATPase, and inhibits 5-hydroxytryptamine accumulation by accelerating leakage of the amine; and that the effects of tributyltin chloride are due to inhibition of the ATPase, and collapse of the transmembrane H⁺-gradient through OH⁻-anion exchange.     

Analogs of adenosine 3',5'-cyclic phosphate. II. Synthesis and enzymatic activity of derivatives of 1,N6-ethenoadenosine 3',5'-cyclic phosphate

The reactions of chloroacetaldehyde with adenosine 3′,5′-cyclic phosphate, and with several analogs modified at C₈ of the purine ring or C₅, of the sugar, lead to the corresponding 1,N⁶-etheno derivatives^d. Similar reactions using other 2-bromoaldehydes or phenacyl bromide give 1,N⁶-ethenonucleotides substituted at the α- or β-positions of the etheno bridge respectively. The ability of these compounds to activate the protein kinases from rabbit muscle and calf brain has been evaluated over a wide range of concentrations. While no derivative proved to be more active than adenosine 3′,5′-cyclic phosphate itself using the enzyme from rabbit muscle, a wide spectrum of activities was found using that from calf brain.     

Kinetics of Hormone-induced H Excretion

A study has been made of the kinetics of hormone-induced H⁺ excretion from peeled Avena coleoptile sections using a new, simple technique involving direct application of the pH electrode to the surface of the section. Hormone-induced H⁺ excretion begins after lags and occurs at rates which are consistent with a role of H⁺ in regulating cell elongation. With fusicoccin, H⁺ excretion begins within the 1st minute, and an external pH of 5 (optimal for wall loosening) is reached in 5 to 8 minutes, while with auxin the lag averages 14 minutes and pH 5 is reached in 20 to 30 minutes. KCN, which inhibits cell elongation in 3 to 5 minutes, stops H⁺ excretion in less than 1 minutes, whereupon the external pH rises sharply. Cycloheximide stops auxin-induced H⁺ excretion in 3 to 8 minutes, and the pH then rises slowly. In the absence of hormones, the pH of the extracellular solution comes to equilibrium at 5.6, but the actual pH of the wall solution is probably about 0.3 unit below this due to Donnan effects.     

Effects of colchicine on the accumulation of vacuolar H+-pyrophosphatase and H+-ATPase in germinating Acacia mangium seeds and the recovery effects by sucrose, indole butyric acid and 6-benzyladenine

Plant vacuoles were isolated from cotyledons of germinatingAcacia mangium seeds, which had been treated with or withoutcolchicine, to measure vacuolar membrane pyrophosphate (PPi)- andATP-dependent H⁺ transport activities, and enzymaticactivities of H⁺-pyrophosphatase(H⁺-PPase) and H⁺-ATPase. Innon-colchicine-treated seeds, activities of the two enzymes increasedrapidly after seed germination to almost a maximal level on the seventhday. A linear function relationship exists in magnitude between PPi- orATP-dependent H⁺transport activity and its correspondingenzymatic activity. The former regression equation is: PPi-dependentH⁺ transport activity(%A.min^–1.g^–1) =–0.039 + H⁺-PPase activity(units.mg^–1) × 1.574, the latter is:ATP-dependent H⁺ transport activity(%A.min^–1.g^–1) =–0.003 + H⁺-ATPase activity(units.mg^–1) × 0.549. In colchicine-treatedseeds, activities of the two enzymes increased very slowly during 8 daysof germination and the relationship to their respectiveH⁺ transport activities was not in agreement with theabove-mentioned regression equations. PPi- and ATP-dependentH⁺ transport activities were lower than thecorresponding values calculated from H⁺-PPase activityand H⁺-ATPase activity according to the two regressionequations, respectively. However, when sucrose, indole butyric acid(IBA), or 6-benzyladenine (6-BA) were applied exogenously to the seedsfollowing colchicine treatment for 3 days, activities ofH⁺-PPase, H⁺-ATPase, PPi- andATP-dependent H⁺ transport in the 6-day-old seedlingsall increased. By statistical analysis, it was concluded that colchicineinhibits cotyledon vacuolar membrane H⁺-PPase,H⁺-ATPase activities, PPi- and ATP-dependentH⁺ transport activities during seed germination andearly seedling growth of Acacia mangium. The inhibitory effectsof colchicine could be overcome by IBA, 6-BA and sucrose to varyingdegrees.     

Concurrent development of resistance to 6-azauridine and adenosine in a mouse cell line

A variant of the hypoxanthine-guanine phosphoribosyltransferase deficient, and adenine phosphoribosyltransferase deficient mouse A9 cell line has been obtained by selecting cells which are resistant to 6-azauridine. These cells are not only resistant to 6-azauridine (5 × 10⁻⁴ M), but also to adenosine (10⁻³ M). Resistance persists indefinitely even in the absence of both compounds. The resistant cells are killed by 5-fluorouridine (10⁻⁶ M), indicating that the part of the salvage pathway for pyrimidine ribonucleotide biosynthesis which is relevant to the action of 6-azauridine is intact. The heritable change producing concurrent resistance to 6-azauridine and adenosine probably involves the de novo pyrimidine biosynthetic pathway.