Expansion of the cellular content of ribonucleoside triphosphates induces cell shrinkage and KCl loss in Ehrlich ascites tumor cells and in Chinese hamster ovary cells.

The conversion to corresponding triphosphate derivatives of various ribonucleosides has been studied in Ehrlich ascites tumor cells and in Chinese hamster ovary cells under conditions that are optimal for cellular uptake of orthophosphate. The initial cellular uptake of orthophosphate is followed by a cellular loss of Cl- which might be consistent with a H2PO4-/Cl- exchange mechanism. Subsequent addition of ribonucleosides to the medium leads to cellular accumulation of the corresponding triphosphate and to a concomitant loss of KCl and to sustained cell volume reduction. The latter two events are quite unspecific with regard to the nucleobase moiety of the ribonucleoside triphosphate accumulated (adenine, guanine and purine being almost equally effective) and they depend in a rather simple way on the increase of the cellular content of these compounds. The KCl loss seems to depend on opening of the separate K+ and Cl- channels. The pharmacological profile of the putative ion channels could not be identified in spite of experiments with conventional blockers. In the case of purine riboside the accumulation of the corresponding triphosphate and concomitant loss of KCl and cell water may be followed by a regain of cell volume due to a continued purine riboside triphosphate accumulation, which apparently depends on the uptake of orthophosphate by cotransport with Na+ and which for osmotic reasons is accompanied by the uptake of water and hence volume increase. The possibility that the nucleoside triphosphate induced opening of a putative Cl- channel may be due to a direct effect of triphosphate on a channel protein is discussed.

Induction of cell surface blebbing by increased cellular Pi concentration.

Cell surface blebbing is an early, reversible characteristic of anoxia described in several different cell types. Blebbing may lead to the formation of large terminal blebs, and ultimately cell death upon rupture of the membrane. In the present work, evidence is presented indicating that Pi may be the immediate mediator of cell surface blebbing: (1) cell surface blebbing can be induced in normoxic Ehrlich ascites tumour cells by a high extracellular concentration of Pp leading to an increase in the cellular Pi concentration; (2) anoxia induces sustained elevation of the cellular Pi concentration and (3) cell surface blebbing during anoxia is reversed upon reoxygenation, and the disappearance of blebbing depends on the decrease in cellular Pi concentration. The rate of disappearance of blebs may be enhanced by the simultaneous addition of adenine and inosine to the growth medium. This leads to a decrease in cellular Pi concentration and to an almost complete restoration of the cellular ATP concentration. It is suggested that Pi is an important mediator of anoxia induced cell damage.

Cell cycle-dependent regulation of cellular ATP concentration, and depolymerization of the interphase microtubular network induced by elevated cellular ATP concentration in whole fibroblasts.

In the present work, evidence is presented indicating that an increased cellular ATP concentration during mitosis may, in conjunction with other factors [Verde et al., 1990: Nature 343:233-238; Andersen et al., 1994: J Cell Biol. 127:1289-1299], induce depolymerization of the interphase microtubular network in cultured fibroblasts. It is shown here that the cellular ATP concentration varies through the cell cycle, reaching a peak at G2M- and minimum at late G1/early S-phase. Furthermore, we have found, using indirect immunofluorescent staining with an antitubulin antibody, that depolymerization of the interphase microtubular network may be induced by increasing the intracellular ATP concentration in cultured fibroblasts from 2.2 mM to 4.1 mM. This may be obtained through addition of adenosine and P1 to the growth medium. Our results indicate that this effect of adenosine and Pi is not mediated via adenosine receptors, but through an elevated cellular ATP concentration. ATP is suggested to act through a concentration-dependent effect on the exchangeable GTP site on tubulin, and not through the action of protein kinases or microtubule-associated proteins.

Net uptake of orthophosphate in Ehrlich ascites tumor cells in the presence of purine riboside may be rate limiting for the expansion of the pool of ribonucleotides.

Incubation with adenosine or with structural analogs thereof may in several cell types under some conditions result in the cellular accumulation of abundant amounts of the corresponding triphosphates. In the present work we have found that incubation of cells at high concentrations of orthophosphate (Pi) results in increased intracellular levels thereof, although they become not as high as the extracellular concentration. In the presence of purine riboside (nebularine, Pr) and high concentration of Pi the intracellular Pi is, however, kept at a low steady-state level, probably because it immediately upon uptake is being trapped primarily as the triphosphate of purine riboside. The latter compound accumulates at a constant rate for at least 1 h. The rate of accumulation of the sum of phosphate residues present in Pi, adenosine phosphates and purine riboside phosphates appears to be proportional to the extracellular concentration of Pi and to be highly dependent on pH (6.5 and 7.0 being optimal and 7.9 nonpermissible) but it is unaffected by substitution of Na+ by choline.

The rate of catabolism of dATP to deoxyadenosine during the growth of different cell lines in vitro.

A method has been developed for the determination of the rate of formation of deoxyadenosine from dATP in cultured cell lines. The lowest rate was found in the T-cell-derived Molt cell line while it was about 70-fold higher in Balb c/3T3 mouse fibroblasts. In the B-cell-derived Raji cells and in the murine sarcoma cell line SEWA it had intermediary values. It is concluded that in some cell types like the 3T3-cells the catabolism of dATP to deoxyadenosine may have a significant regulatory effect on the cellular content of dATP.