Robert M. Wohlhueter

Permeation of Nucleosides, Nucleic Acid Bases, and Nucleotides in Animal Cells

Publisher Summary This chapter discusses the importance of the mechanism of permeation of nucleosides, nucleobases, and nucleotides through the cell membranes of eukaryotes. Some of the reasons of its importance include (1) many anticancer and immunosuppressive agents presently in use or under development are nucleoside, nucleotide, or nucleobase analogs and a clear understanding of their mode of entry into cells and metabolism is important in the assessment of their mode of action, efficacy, and optimal administration, and (2) radioactively labeled nucleosides and nucleic acid bases are widely used as precursors to label specifically the nucleic acids of various types of organisms or of the viruses or plasmids replicating therein as well as to assess the rates of nucleic acid synthesis. An interpretation of the rates of nucleoside and base incorporation into nucleic acids, be it RNA or DNA, depends on a clear understanding of the extent to which these rates may reflect the rates of the conversion of the extracellular substrate to intracellular nucleotides, which are the direct precursors in nucleic acid synthesis. Elimination of the ambiguities inherent in metabolizing cells is of clear advantage to transport studies. Nucleoside and purine transport have been studied successfully in the absence of intracellular metabolism by the use of erythrocytes or of mutant clones of cultured animal cells that are deficient in specific metabolic enzymes and by the use of cell/substrate systems in which substrate metabolism is blocked in some other manner. The chapter also discusses the carrier model for facilitated diffusion and tests for its applicability to nucleoside and base transport.

Laminin Alterations After In Vitro Nonenzymatic Glycosylation

Laminin, a basement membrane protein derived from the matrix of the Engelbreth-Holm-Swarm murine tumor, was nonenzymatically glycosylated in vitro in the presence of increasing glucose concentrations. The amount of glucose incorporated per laminin molecule was shown to be proportional to the molarity of glucose used. Nonenzymatic glycosylation resulted in formation of cross-links and alterations of the cruciform shape of laminin molecules; these alterations were dramatic when high concentrations of glucose were used. One of the functions of laminin, the process of self-assembly, was shown to be impaired after in vitro nonenzymatic glycosylation. Glucose incorporation resulted in a dramatic decrease of long-to-long laminin dimers, which normally form during the initial steps of assembly. Furthermore, nonenzymatic glycosylation of laminin reduced its ability to self-associate into complexes larger than dimers, as judged by turbidimetry. The observed decrease of maximal turbidity was proportional to the degree of nonenzymatic glycosylation. Aminoguanidine, which has been suggested to inhibit cross-link formation, was shown to restore to a large extent the shape of laminin, the percentage of long-to-long arm dimers, and the maximal turbidity when included in the mixtures of laminin and glucose. These data suggest that structural and functional alterations of laminin may be primarily due to formation of crosslinks. Such modifications of laminin (along with our basement membrane components) may contribute to the morphological and physiological changes observed in basement membranes under diabetic conditions.

The roles of transport and phosphorylation in nutrient uptake in cultured animal cells.

Publisher Summary Three main classes of nutrients—nucleosides, nucleobases, and hexoses (and possibly a fourth, the water-soluble vitamins)—are taken up into cultured cells as a result of the tandem action of a nonconcentrative transport system and an intracellular enzyme that introduces into the transported substrate an anionic group thus confering impermeability. For several substrates representing the first three of these nutrient classes, both members of the uptake pathway are characterized kinetically: (1) the transport systems with cells and substrates that, by virtue of enzyme deficiency, adenosine triphosphate (ATP) depletion, or chemical design, are metabolically inert and (2) the enzymes, as purified proteins, in idealized milieu. The consequences of changes in substrate concentration, in temperature, and in the effective kinetic parameters of transport and phosphorylation on rates of uptake demonstrate the complexity of this interdependence and the errors of interpretation that can accrue if the complexity is overlooked. Flux in a tandem pathway follows a biphasic time course. The first phase corresponds to the attainment of steady-state levels of free intracellular substrate. The second phase represents steady-state flux through the pathway—that is, the rate of accumulation of impermeable product.

Chapter 16 A Rapid-Mixing Technique to Measure Transport in Suspended Animal Cells: Applications to Nucleoside Transport in Novikpff Rat Hepatoma Cells

Publisher Summary The chapter discusses techniques, which permit an operational separation of transport and metabolism. This separation can be achieved by genetic, chemical, or kinetic manipulation, or a combination thereof. The transport of various compounds across mammalian cell membranes is frequently found to occur with a rapidity which necessitates collecting data at intervals of a few seconds. By means of a dual-syringe device, suspended cells can be mixed nearly instantaneously with radioactively labeled substrate and separated from the substrate again within seconds by centrifugation into silicone oil. Depending on the cell-substrate system under investigation, initial transport velocities may be either measured directly or calculated from the time course with which equilibrium across the membrane is attained. With nonmetabolizing systems, the dual-syringe apparatus is adaptable to a variety of experimental protocols-zero-trans, equilibrium exchange, and infinite-cis—which in combination make possible a thorough kinetic characterization of a transport system.

Nucleoside transport in cultured mammalian cells multiple forms with different sensitivity to inhibition by nitrobenzylthioinosine or hypoxanthine

The zero-trans influx of 500 microM uridine by CHO, P388, L1210 and L929 cells was inhibited by nitrobenzylthioinosine ( NBTI ) in a biphasic manner; 60-70% of total uridine influx by CHO cells and about 90% of that in P388, L1210 and L929 cells was inhibited by nmolar concentrations of NBTI (ID50 = 3-10 nM) and is designated NBTI -sensitive transport. The residual transport activity, designated NBTI -resistant transport, was inhibited by NBTI only at concentrations above 1 microM (ID50 = 10-50 microM). S49 cells exhibited only NBTI -sensitive uridine transport, whereas Novikoff cells exhibited only NBTI -resistant uridine transport. In all instances NBTI -sensitive transport correlated with the presence of between 7 7 X 10(4) and 7 X 10(5) high-affinity NBTI binding sites/cell (Kd = 0.3-1 nM). Novikoff cells lacked such sites. The two types of nucleoside transport, NBTI -resistant and NBTI -sensitive, were indistinguishable in substrate affinity, temperature dependence, substrate specificity, inhibition by structurally unrelated substances, such as dipyridamole or papaverine, and inhibition by sulfhydryl reagents or hypoxanthine. We suggest, therefore, that a single nucleoside transporter can exist in an NBTI -sensitive and an NBTI -resistant form depending on its disposition in the plasma membrane. The sensitive form expresses a high-affinity NBTI binding site(s) which is probably made up of the substrate binding site plus a hydrophobic region which interacts with the lipophilic nitrobenzyl group of NBTI . The latter site seems to be unavailable in NBTI -resistant transporters. The proportion of NBTI -resistant and sensitive uridine transport was constant during proportion of NBTI -resistant and sensitive uridine transport was constant during progression of P388 cells through the cell cycle and independent of the growth stage of the cells in culture. There were additional differences in uridine transport between cell lines which, however, did not correlate with NBTI sensitivity and might be related to the species origin of the cells. Uridine transport in Novikoff cells was more sensitive to inhibition by dipyridamole and papaverine than that in all other cell lines tested, whereas uridine transport in CHO cells was the most sensitive to inactivation by sulfhydryl reagents.

Properties of the thymidine transport system of chinese hamster ovary cells as probed by nitrobenzylthioinosine

SummaryThe transport of thymidine into Chinese hamster ovary cells grown in suspension culture was measured under conditions in which thymidine was not metabolized, namely, when cells had been depleted of ATP. The system transporting thymidine was saturable (Kmzt=70μM), rapid (50% of transmembrane equilibrium level attained within 8 sec), and was apparently shared by other nucleosides, but not thymine or hypoxanthine. 6([4-nitrobenzyl]thio)-9-β-d-ribofuranosylpurine, “nitrobenzylthioinosine”, inhibited thymidine transport in a simple, noncompetitive fashion with an apparentKi=1.0 nM (based on total concentration of inhibitor, which significantly overestimates that of free inhibitor). The rate of expression of inhibition was slow (t1/2=17 sec) relative to the rate of association of thymidine with its transporter, and thymidine partially protected the transport system against inhibition by nitrobenzylthioinosine. The dissociation constant for the inhibitortransporter complex was estimated at about 0.1 nM, and the number of binding sites per cell at about 6×104. HeLa, P388 murine leukemia, and mouse L cells were as sensitive to nitrobenzylthioinosine inhibition of thymidine transport as Chinese hamster ovary cells; Novikoff rat hepatoma cells were much less sensitive.

Nitrobenzylthioinosine-sensitive and -resistant nucleoside transport in normal and transformed rat cells

Cultured Novikoff rat hepatoma and Walker 256 carcinoma cells have previously been reported to express only nitrobenzylthioinosine (NBTI)-resistant uridine transport and to lack high affinity NBTI-binding sites, whereas the latter are common on all other types of cultured mammalian cells from different species [1-7) X 10(5) sites/cell) which have been investigated with the exception of a transport-deficient cell variant which lacks high-affinity NBTI-binding sites. The present study shows that lack of NBTI sensitivity of transport and of NBTI-binding sites in Novikoff and Walker 256 cells are not related to the species or tissue origin of these cells. Uridine transport in a variant (NRM) of Novikoff hepatoma cells, in HTC rat hepatoma cells, normal rat kidney (NRK) cells, rat erythrocytes and rat hepatocytes was inhibited 15-60% by 10-500 nM NBTI and the cells expressed high-affinity NBTI-binding sites (Kd = 0.1-0.6 nM). The apparent turnover numbers for the NBTI-sensitive nucleoside carriers fell into two classes, with those for transformed cells about 10-times higher than those for the normal rat cells.

Inhibition of the transport of adenosine, other nucleosides and hypoxanthine in Novikoff rat hepatoma cells by methylxanthines, papaverine, N6-cyclohexyladenosine and N6-phenylisopropyladenosine

Theophylline, caffeine, isobutylmethylxanthine, papaverine, N6-cyclohexyladenosine, N6-allyl-N6-cyclohexyladenosine ( ACHA ) and N6-L-phenylisopropyladenosine (L-PIA) inhibited the transport of adenosine, uridine and hypoxanthine in Novikoff rat hepatoma cells. The IC50 values for the inhibition of uridine transport ranged from 5 microM for ACHA to 3200 microM for caffeine and were inversely proportional to the lipid solubility of the inhibitors. L-PIA and papaverine inhibited uridine influx in a non-competitive manner, having a greater influence on the Michaelis-Menten constant than on maximum velocity, just as observed previously for the inhibition of nucleoside transport by dipyridamole and hypoxanthine. [3H]L-PIA rapidly accumulated in Novikoff cells at 25 degrees to about five times higher levels than present extracellularly. The initial rates of L-PIA uptake were directly proportional to its extracellular concentration between 0.01 and 240 microM and not affected by structurally related analogs, methylxanthines, papaverine, dipyridamole, or 2 mM uridine, but were dependent on temperature. We conclude that L-PIA inhibits nucleoside transport in these cells without being significantly transported by the carrier, that it equilibrates rapidly across the plasma membrane without carrier mediation consistent with its lipophilicity, and that it accumulates concentratively in cells due to partitioning into membrane lipids and binding to intracellular components.

On the functional symmetry of nucleoside transport in mammalian cells

The transport of uridine and thymidine has been examined in HeLa cells, in Novikoff rat hepatoma cells and in human erythrocytes, with the purpose of comparing influx, efflux and isotopic exchange at chemical equilibrium. The results support the following conclusions: (i) In all three cell types influx and efflux are comparable; (ii) HeLa and Novikoff cells show no trans-effect, while erythrocytes show a 5-fold trans-stimulation; (iii) a single kinetic entity accounts for nucleoside transport in HeLa and Novikoff cells - no parallel routes of permeation with Km less than 40 microM were detected. For the cultured cells, the flux data conform to the kinetic model of a single, carrier-mediated transport system symmetrical with respect to direction, and with equal mobilities of substrate-loaded and empty carrier.

Characterization of immunologically active cyanogen bromide peptide fragments of bovine and human retinal S-antigen *

Peptides which account for most, if not all, of the cyanogen bromide (CNBr)-generated peptide fragments of bovine retinal S-antigen have been identified and examined for their immunoreactivity with antisera raised to bovine and human S-antigen and with immune lymphocytes further selected twice in vitro with either bovine or human S-antigen. Amino-acid sequencing of a large fragment of S-antigen missing a small N-terminal peptide revealed the location of three overlapping CNBr peptides near the N-terminus. Amino-acid sequencing of several other CNBr peptides has allowed their position in a partial DNA-predicted sequence of the carboxy terminal half of the antigen to be determined. The total CNBr digest of human S-antigen was also prepared and compared with the fragments of the bovine antigen. Sera from rats immunized with bovine or human S-antigen were similar in their specificity in the enzyme-linked immunosorbent assay (ELISA) for purified bovine peptides except for the CB21 peptide which was not significantly bound by anti-human S-antigen sera. All of the other bovine peptides recognized by anti-bovine S-antigen sera were also bound by antibodies in the sera raised to the human antigen. The CNBr peptides of human and bovine S-antigen were extracted from gel slices and also assayed in the ELISA. Peptides of bovine S-antigen purified by HPLC were tested for their ability to stimulate an in vitro proliferative response in lymphocytes from Lewis rats immunized with either bovine or human S-antigen. Only quantitative differences in the proliferative response to human vs. bovine S-antigen and CNBr peptides were found. Methodology for the purification and analysis of the peptides is presented as well as the properties of the peptides.