Membrane Transport of Nucleoside Analogues in Mammalian Cells

Abstract

This paper attempts to summarize what is known, from rapid kinetic studies of cell membrane transport, about the mechanism by which nucleoside analogues permeate the plasma membrane of mammalian cells.     

Abscisic Acid Transport in Human Erythrocytes

Abscisic acid (ABA) is a plant hormone involved in the response to environmental stress. Recently, ABA has been shown to be present and active also in mammals, where it stimulates the functional activity of innate immune cells, of mesenchymal and hemopoietic stem cells, and insulin-releasing pancreatic β-cells. LANCL2, the ABA receptor in mammalian cells, is a peripheral membrane protein that localizes at the intracellular side of the plasma membrane. Here we investigated the mechanism enabling ABA transport across the plasmamembrane of human red blood cells (RBC). Both influx and efflux of [³H]ABA occur across intact RBC, as detected by radiometric and chromatographic methods. ABA binds specifically to Band 3 (the RBC anion transporter), as determined by labeling of RBC membranes with biotinylated ABA. Proteoliposomes reconstituted with human purified Band 3 transport [³H]ABA and [³⁵S]sulfate, and ABA transport is sensitive to the specific Band 3 inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid. Once inside RBC, ABA stimulates ATP release through the LANCL2-mediated activation of adenylate cyclase. As ATP released from RBC is known to exert a vasodilator response, these results suggest a role for plasma ABA in the regulation of vascular tone.     

Inhibition of Cation Channels in Human Erythrocytes by Spermine

In erythrocytes, spermine concentration decreases gradually with age, which is paralleled by increases of cytosolic Ca2+ concentration, with subsequent cell shrinkage and cell membrane scrambling. Cytosolic Ca2+ was estimated from fluo-3 fluorescence, cell volume from forward scatter, cell membrane scrambling from annexin V binding and cation channel activity with whole-cell patch-clamp in human erythrocytes. Extracellular spermine exerted a dual effect on erythrocyte survival. At 200 μM spermine blunted the increase of intracellular Ca2+, cell shrinkage and annexin V binding following 48 h exposure of cells at +37 °C. In contrast, short exposure (10-30 min) of cells to 2 mM spermine was accompanied by increased cytosolic Ca2+ and annexin binding. Intracellular addition of spermine at subphysiological concentration (0.2 μM) significantly decreased the conductance of monovalent cations (Na+, K+, NMDG+) and of Ca2+. Moreover, spermine (0.2 μM) blunted the stimulation of voltage-independent cation channels by Cl? removal. Spermine (0.2 and 200 μM) added to the extracellular bath solution similarly inhibited the cation conductance in Cl?-containing bath solution. The effect of 0.2 μM spermine, but not the effect of 200 μM, was rapidly reversible. Acute addition (250 μM) of a naphthyl acetyl derivative of spermine (200 μM) again significantly decreased basal cation conductance in NaCl bath solution and inhibited voltage-independent cation channels. Spermine is a powerful regulator of erythrocyte cation channel cytosolic Ca2+ activity and, thus, cell survival.     

The Role of Oxidized Nicotinamide Adenine Dinucleotide in Fluoride Inhibition of Active Sodium Transport in Human Erythrocytes

The rate coefficient for ²²Na release from previously labeled human erythrocytes was determined in the presence of 0.1–10 mM sodium fluoride (F). The oxidized nicotinamide adenine dinucleotide (NAD⁺) level at the end of 2 hr of incubation in tris(hydroxymethyl)aminomethane (Tris)-Ringer medium was also measured. Both parameters decreased proportionately as F concentration was raised. Both F-induced changes were immediate and were reversed by 10 mM pyruvate. The decrease in NAD⁺ concentration following enolase inhibition by F is attributed to a diminished rate of formation in the reaction catalyzed by lactic dehydrogenase (LDH) with undiminished continued utilization in the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It is postulated that the NAD⁺ lowering limited the GAPDH step, resulting in proportionate decreases in the rates of phosphoglycerate kinase (PGK) and Na,K-dependent adenosine triphosphatase (Na,K-ATPase), a reaction sequence thought to link glycolysis with active Na extrusion. Adding pyruvate with F increased NAD⁺ production at the LDH step, thus reactivating GAPDH, PGK, and Na,K-ATPase and leading to the observed restoration of ²²Na release. The results suggest, therefore, that F inhibits active Na transport in intact human erythrocytes indirectly through a lowering of NAD⁺, although, direct inhibition of the Na,K-ATPase by F may possibly occur simultaneously.     

Steady-State, Hemoglobin-Facilitated O2 Transport in Human Erythrocytes

We measured the rate of oxygen transport through thin (165 µ) films of packed erythrocytes (Hb concentration = 30 g/100 ml). Under optimal conditions steady-state O₂ diffusion was nearly three times that found when the hemoglobin was prevented from acting as a carrier molecule by carbon monoxide binding or high oxygen back pressure. After each experiment we measured hemolysis and found that it averaged less than 1%. Hemolysis could not account for the facilitation, thus proving that facilitated transport of O₂ by hemoglobin can occur in red blood cells. The rate of facilitated transport was identical for Hb solutions of equal concentration to the cells. We interpret this to mean that under the conditions of our experiments the red cell membrane offers no detectable diffusion resistance to O₂ and that the mobility of Hb in intact red cells is the same as in concentrated Hb solution.     

Antiviral Activity of Selected Nucleoside Analogues Against Human Herpes Virus Type 6

Abstract

HHV-6 was evaluated in vitro for its susceptibility to a broad range of nucleoside analogues. PFA and several acyclic nucleoside phophonates emerged as the most potent inhibitors of HHV-6 replication as monitored by a newly developed immunofluorescence / flow cytometric assay as well as by microscopical evaluation of their inhibitory effect on HHV-6-induced cytopathogenicity.     

Correlation of Growth Inhibition Patterns to Nucleoside Transport Models in Neurospora crassa

Growth of inhibition patterns provide evidence for a common nucleoside transport or utilization system, a separate system or systems for adenine transport, and another adaptable mechanism of adenosine transport.     

Synthesis of New Thiazolidinone Nucleoside Analogues

Abstract

The synthesis of new thiazolididone nucleoside analogues is described. Among the different proposed synthetic pathways, the condensation of various nucleic bases using TMSOTf and Et₃N as coupling reagents on a key sulfoxide thiazolidinone intermediate led to the desired compounds in a one-pot procedure. Analytical data and NMR studies confirmed the proposed structure assignment for these compounds.     

Synthesis of cis-Disubstituted Cyclobutenyl Nucleoside Analogues

Abstract

cis-Disubstituted cyclobutene nucleosides analogues were prepared by a linear synthesis starting from cis-cyclobutene dicarboxylic anhydride. This strategy involved mild reaction conditions with intent to restrict the thermal electrocyclic ring opening into (Z,E)-dienes.     

力学因素对人红细胞葡萄糖运输和阴离子交换的影响

机械应力在心血管系统的正常生理和病理中都起着重要的作用。实验中观察到当提高红细胞悬浮液的旋转速度时会导致葡萄糖跨膜输入的速率增加。改变溶液渗透压及用使细胞膜曲率变化的药物（氯丙嗪）是对红细胞作用的另二种力学因素。研究发现它们同样也能对葡萄糖和阴离子的运输有影响,根据运输速率的温度特性给出了这些力学因素作用下葡萄糖、阴离子运输时活化能的变化,活化能的减小和运输速率的增加有很好的对应关系;活化能减小使膜上运输蛋白在运输过程中的构象变化更为容易,对红细胞血影的内禀荧光淬灭测量量表明,机械应力是通过影响膜上葡萄糖运输蛋白（GLUT1）和阴离子交换蛋白（带3蛋白）物构象起作用的,当用抑制剂抑制了阴离子的运输后。观察到此时葡萄糖跨膜运输对机械应力的响应发生改变,这再次表明在红细胞膜上GLUT1和带3蛋白之间存在着信号连接。     

The Kinetics of Selective Biological Transport: II. Equations for Induced Uphill Transport of Sugars in Human Erythrocytes

Equations describing the movement of sugars during induced uphill transport were derived on the assumption of a simple carrier transport mechanism and subjected to experimental verification. Since there was good agreement between the experimental points and the theoretical curves, no changes in the original postulates were required.     

The Kinetics of Selective Biological Transport: I. Determination of Transport Constants for Sugar Movements in Human Erythrocytes

A method for the determination of the affinity constants and transfer rate constants of an equalizing selective transport system is derived from the simple transport mechanism, and applied to monosaccharide movements in human red cells. It is similar to the method of Widdas but does not require his approximations. Results are compared with those of the other workers.     

Structure-Activity Relationship of Pyrimidine Heterosubstituted Nucleoside Analogues

Abstract

The structure-activity relationship of sixteen 3-deaza, C-4 substituted pyrimidines and imidazo[1,2-c]pyrimidine bases of 1,3-oxathiolanes and 1,3-dioxolanes revealed good anti-HBV activity in 2.2.15 cells transfected with human hepatitis B virus of the imidazo[1,2-c]pyrimidine nucleosides 21, 25 and 29. Two procedures for the preparation of C-4 substituted analogues are reported based on nucleophilic displacement of a sulfonamide or imidazole by a variety of nitrogen nucleophiles.     

Synthesis of Nucleoside Analogues Using Immobilised N-Deoxyribosyltransferases

Crude N-deoxyribosyltransferase from Lactobacillus leichmannii was immobilised by hydrophobic interaction on octyl-Sepharose or by covalent attachment to poly(acrylamide-co-N-acryl-oxysuccinimide) (PAN). Little enzyme activity was retained by entrapment in K-carrageenan. N-deoxyribosyltransferase immobilised on PAN was used to prepare 2'-deoxy-2-thiouridine.     

Novel Types of Spin Labelled Nucleoside Analogues

Abstract

A variety of modified nucleosides or dinucleosides bearing one of the following functions have been prepared: N-hydroxyureas, N-hydroxyamines, N-hydroxycarbamates, α-(N-hydroxyamino)phosphonates. Upon oxidation, these compounds afford the corresponding aminoxyl free radicals which have been studied by EPR spectroscopy. Some of these compounds exhibited antiviral properties.     

The Binding Specificity of Amino Acid Transport System y+L in Human Erythrocytes is Altered by Monovalent Cations

System y⁺L is a broad-scope amino acid transporter which binds and translocates cationic and neutral amino acids. Na⁺ replacement with K⁺ does not affect lysine transport, but markedly decreases the affinity of the transporter for l-leucine and l-glutamine. This observation suggests that the specificity of system y⁺L varies depending on the ionic composition of the medium. Here we have studied the interaction of the carrier with various amino acids in the presence of Na⁺, K⁺, Li⁺ and guanidinium ion. In agreement with the prediction, the specificity of system y⁺L was altered by the monovalent cations. In the presence of Na⁺, l-leucine was the neutral amino acid that interacted more powerfully. Elongation of the side chain (glycine - l-norleucine) strengthened binding. In contrast, bulkiness at the level of the β carbon was detrimental. In K⁺, the carrier behaved as a cationic amino acid specific carrier, interacting weakly with neutral amino acids. Li⁺ was found to potentiate neutral amino acid binding and in general the apparent affinities were higher than in Na⁺; elongation of the nonpolar side chain made a more important contribution to binding and the carrier was more tolerant towards β carbon substitution. Guanidinium stimulated the interaction of the carrier with neutral amino acids, but the effect was restricted to certain analogues (e.g., l-leucine, l-glutamine, l-methionine). Thus, in the presence of guanidinium, the carrier discriminates sharply among different neutral amino acids. The results suggest that the monovalent cations stabilize different carrier conformations. Received: 22 January 1996/Revised: 26 April 1996     

The Convenient Synthesis of Unsaturated Nucleoside Analogues in Water under Microwave Irradiation

A convenient method for the regioselective synthesis of unsaturated nucleoside analogs in water under microwave irradiation was developed. All pyrimidine and purine nucleoside derivatives were exclusively alkylated at N1 and N9 respectively in good to excellent yields. In addition, this system could tolerate a broad range of functional groups, such as chloro, bromo, iodo, alkyl, amino, and hydroxyl groups. More importantly, the reaction scale could be enlarged to 50 mmol which made this route attractive for industrial application.     

Synthesis of New Fluorescent Nucleoside Analogues and Application to the Study of Human Deoxycytidine Kinase

Abstract

We have determined the affinity of human deoxycytidine kinase with respect to new fluorescent N-methylanthraniloyl cytidine derivatives or non fluorescent enantiomeric cytidine analogues. New results regarding the enantioselectivity and the mechanism of the enzyme are presented.     

Synthesis of Novel Olefinic Carbocyclic Purine Nucleoside Analogues

Abstract

The synthesis of optically pure unsaturated carbocyclic nucleoside analogues is described. (3,4S)-Bis(t-butyldiphenyl silyloxymethyl)-1R and 1S cyclopent-2-en-1-ol were coupled with 6-chloropurine and 2-amino-6-chloropurine respectively, using a modified Mitsunobu reaction. The products were reacted further using standard procedures to give compounds 12, 14, 16 and 18.