Cell cycle and growth stage-dependent changes in the transport of nucleosides, hypoxanthine, choline, and deoxyglucose in cultured Novikoff rat hepatoma cells.

Populations of Novikoff rat hepatoma cells (subline N1S1-67) were monitored for the rates of transport of various substrates and for their incorporation into acid-insoluble material as a function of the age of cultures of randomly growing cells in suspension as well as during traverse of the cells through the cell cycle. Populations of cells were synchronized by a double hydroxyurea block or by successive treatment with hydroxyurea and Colcemid. Kinetic analyses showed that changes in transport rates related to the age of cultures or the cell cycle stage reflecte alterations in the V max of the transport processes, whereas the Km remained constant, indicating that changes in transport rates reflect alterations in the number of functional transport sites. The transport sites for uridine and 2-deoxy-D-glucose increased continuously during traverse of the cells through the cell cycle, whereas those for choline and hypoxanthine were formed early in the cell cycle. Increases in thymidine transport sites were confined to the S phase. Synchronized cells deprived of serum failed to exhibit normal increases in transport sites, although the cells divided normally at the end of the cell cycle. Arrest of the cells in mitosis by treatment with Colcemid prevented any further increases in transport rates. The formation of functional transport sites was also dependent on de novo synthesis of RNA and protein. Inhibition of DNA synthesis in early S phase inhibited the increase in thymidine transport rates which normally occurs during the S phase, but had no effect on the formation of the other transport systems. Transport rates also fluctuated markedly with the age of the cultures of randomly growing cells, reaching maximum levels in the mid-exponential phase of growth. The transport systems for thymidine and uridine were rapidly lost upon inhibition of protein and RNA synthesis, and thus seem to be metabolically unstable, whereas the transport systems for choline and 2-deoxy-D-glucose were stable under the same conditions.

Facilitated transport of 6-mercaptopurine and 6-thioguanine and non-mediated permeation of 8-azaguanine in Novikoff rat hepatoma cells and relationship to intracellular phosphoribosylation.

6-Mercaptopurine and 6-thioguanine strongly inhibited the zero-trans entry of hypoxanthine into Novikoff rat hepatoma cells which lacked hypoxanthine/guanine phosphoribosyltransferase, whereas 8-azaguanine had no significant effect. 6-Mercaptopurine was transported by the hypoxanthine carrier with about the same efficiency as its natural substrates (Michaelis-Menten constant = 372 +/- 23 microM; maximum velocity = 30 +/- 0.7 pmol/microl cell H2O per s). 8-Azaguanine entry into the cells, on the other hand, showed no sign of saturability and was not significantly affected by substrates of the hypoxanthine/guanine carrier. The rate of entry of 8-azaguanine at 10-100 microM amounted to only about 5% of that of hypoxanthine transport and was related to its lipid solubility in the same manner as observed for various substances whose permeation through the plasma membrane is believed to be non-mediated. Only the non-ionized form of 8-azaguanine (pKa = 6.6) permeated the cell membrane. Studies with wild type Novikoff cells showed that permeation into the cell was the main rate-determining step in the conversion of extracellular 8-azaguanine to intracellular aza-GTP and its incorporation into nucleic acids. In contrast, 6-mercaptopurine was rapidly transported into cells and phosphoribosylated; the main rate-determining step in its incorporation into nucleic acids was the further conversion of 6-mercaptopurine riboside 5'-monophosphate.

Effect of irradiation on glycosaminoglycans content in rat tissue.

Glycosaminoglycan (GAGs) fractions were determined in tissues (skin, liver, lungs, aortic wall) and blood serum of rats irradiated with a single dose of 500 R. An increase of total GAGs as well as changes in the fractions were found in the tissues and urine of exposed rats.

[Restoration of the left atrial mechanical function after successful electrical cardioversion]. / Powrót czynnosci mechanicznej lewego przedsionka po skutecznej kardiowersji elektrycznej.

Some papers indicate that restoration of the left atrial mechanical function (LAMF) can be delayed to even 7 days after successful electrical cardioversion. The goals of the paper include the estimation of delay in restoration of LAMF and factors that influence it. 75 patients (53 males and 22 females, av. age 56.5 +/- 10.8) after elective electrical cardioversion underwent daily echocardiography to determine LAMF, taking A/E ratio > 1/3 as a criterion. The patients were divided into 2 subgroups: G1--with restoration of LAMF < 24 h after cardioversion and G2: the remaining ones. The analysed parameters were: left atrium diameter, rheumatic valvular disease, duration of atrial fibrillation, global and segmental contractility disorders. The data were analysed with ANOVA. The restoration of LAMF < 24 h was found in 53 patients; on the 2nd day in 6 patients, on the 3rd-8th day in 14 patients. In 2 patients no LAMF could be found on the 14th day after cardioversion. Failure to restore LAMF on the cardioversion day was related to: left atrium enlargement, rheumatic valvular disease, duration of atrial fibrillation > 14 days and contractility disorders.

Purine and pyrimidine transport by cultured Novikoff cells. Specificities and mechanism of transport and relationship to phosphoribosylation.

Adenine, guanine, and hypoxanthine were rapidly incorporated into the acid-soluble nucleotide pool and nucleic acids by wild type Novikoff cells. Incorporation followed normal Michaelis-Menten kinetics, but the following evidence indicates that specific transport processes precede the phosphoribosyltransferase reactions and are the rate-limiting step in purine incorporation by whole cells. Cells of an azaguanine-resistant subline of Novikoff cells which lacked hypoxanthine-guanine phosphoribosyltransferase activity and failed to incorporate guanine or hypoxanthine into the nucleotide pool, exhibited uptake of guanine and hypoxanthine by a saturable process. Similarly, wild type cells which had been preincubated in a glucose-free basal medium containing KCN and iodoacetate transported guanine and hypoxanthine normally, although a conversion of these purines to nucleotides did not occur in these cells. The mutant and KCN-iodoacetate treated wild type cells also exhibited countertransport of guanine and hypoxanthine when preloaded with various purines, uracil, and pyrimidine nucleosides. The cells also possess a saturable transport system for uracil although they lack phosphoribosyltransferase activity for uracil. In the absence of phosphoribosylation, none of the substrates was accumulated against a concentration gradient. Thus transport is by facilitated diffusion (nonconcentrative transport). Furthermore, the apparent Km values for purine uptake by untreated wild type and azaguanine-resistant cells were higher and the apparent Vmax values were lower than those for the corresponding phosphoribosyltransferases...

Membrane effects of cytochalasin B. Competitive inhibition of facilitated diffusion processes in rat hepatoma cells and other cell lines and effect on formation of functional transport sites.

Cytochalasin B competitively inhibits the transport of 2-deoxy-D-glucose and thymidine in a number of different cell lines (Novikoff rat hepatoma cells, mouse L, S180 and Ki-MSV-transformed BALB/3T3 cells, and human HeLa cells). The apparent Km values for the transport of these substrates as well as the apparent Ki values for the inhibition by cytochalasin B are very similar for the various cell lines, and the effect is readily and completely reversed by removal of the chemical. Thymidine transport by Chinese hamster ovary cells however, is little affected by cytochalasin B, whereas the transport of 2-deoxy-D-glucose, uridine and guanine by these cells is competitively inhibited to about the same extent as in other cell lines. In addition and concomitant with the inhibition of cytokinesis and an alteration in cell shape, cytochalasin B also impairs and delays the formation of functional transport sites for thymidine, guanine and choline in synchronized populations of Novikoff cells without affecting the apparent affinities of the transport systems for their substrates. This effect is unrelated to the direct inhibition of the transport processes, since the drug does not directly inhibit choline transport and has no effect on the formation of 2-deoxy-D-glucose transport sites in spite of the fact that it strongly inhibits the transport of this substrate. The inhibition of functional transport sites may be due to the induction of a structural alteration in the membrane by cytochalasin B which impairs the insertion of new proteins of certain but not all transport systems into the membrane.

G2+M arrest of cultured mammalian cells after incorporation of tritium-labeled nucleosides.

Novikoff rat hepatoma cells were propagated in suspension cultures containing 0.5 to 10 muC of 3H-methyl-thymidine, 3H-5-uridine, 3H-G-adenosine or 3H-8-adenine. The presence of the 3H-labeled precursors caused an inhibition of cell replication which was due to a delay or arrest of the cells in G2 and M. The degree of inhibition was proportional to the amount of radioactivity incorporated into nucleic acids. Almost immediate and complete inhibition resulted from incubation with 10 muC 3H-thymidine/ml. The presence of 0.5 muC 3H-thmidine/ml caused a significant increase in the relative proportion of cells in G2+M, even though the population doubling time of the culture appeared to be unaltered.