(Received Septcmber 1 Deccmbcr 7, 19x2) -EJH 59591. The ability of external ATP to induce calcium uptake in isolated rat liver cells was further characterized. Stimulation of calcium uptake was specific for ATP, other nucleotides or ATP metabolites had no comparable effect. ATP was dephosphorylated while stimulating calcium uptake, but there was no stoichiometry between ATP hydrolysis and calcium uptake nor did dephosphorylation depend on calcium concentration. ATP acted from outside and was dephosphorylated by an ccto-ATPase of the cells.2. In addition to its direct action, ATP enhanccd succinate-dependent calcium uptake in a coopcrativc fashion. This is best explained by different sites of action. ATP increases cell membrane permeability while succinatc stimulates uptake into mitochondria.3. ATP was able to lower Na' and K t gradients and the pH gradient between cells and incubation medium. Increasing calcium concentration countcracted this effect though calcium uptake was then stimulated.4. Succinate alone did not affect monovalent cation gradients but raised the pH gradient. It partially coiinteractcd the ATP effects on these gradients.5. Since catecholamine-like actions of ATP may be mediated by an increase in cytoplasmic calcium concentration, the action of extracellular ATP can be taken as a model t o study the role of calcium as a transmitter of hormone actions. From interdependence between ATP-stimulated and succinatc-stimulated calcium uptake, conclusions can be drawn on the resulting cytoplasmic calcium concentration and its effect on plasma membrane permeability.In a previous study, isolated liver cells proved to be a useful model to study the delicately poised equilibrium of calcium distribution across the various cellular compartments [l]. This was especially true for disclosing the role of cellular mitochondria in this interplay. Respiratory substrates, pH, inorganic phosphate, and the redox grade of pyridine nucleotides were found important in triggering calcium movements at the mitochondria1 site, whereas external ATP and La3+ induced calcium uptake at the plasma membrane site [I. 21.In this report, thc action of added ATP is further characterizcd. The responses of functional parameters such as cation distribution and the pH gradient between cells and incubation medium are compared with those seen after succinate addition. Relations between the different responses and the probablc changes in cytoplasmic calcium concentrations are discussed. This is of interest because external ATP was previously reported to exert a catecholamine-like action on the plasma membrane permeability [3, 41 and to prcvent the stimulatory effect of insulin on hexose transport and oxidation [S -71. As r-adrenergic stimulation generally results in changes in both calcium and potassium distribution (for review see [8]) the study of ATP action might shed light on the relcvance of various cellular parameters in a state similar to that during a-adrenergic action. MATERIALS AND METHODS Radiochmicals.
Seedlings of Mutthiolu incunu (crucifer) are able to take up exogenous homologous DNA by the roots. DNA homogenously labelled with [3H]adenine and 5-bromodeoxyuridine is incorporated into the plants in a macromolecular form. Intact donor DNA and a fraction with a buoyant density intermediate between that of the donor and the recipient DNA can be recovered. Analysis of this intermediate fraction by ultrasonication and alkali treatment allows the suggestion that homologous DNA is integrated as a double-stranded DNA which becomes covalently linked to the recipient DNA.Control experiments in which seedlings were incubated in a mixture simulating donor DNA degradation products in the presence and absence of unlabelled competitors suggest that these results are not due to the breakdown of donor DNA and reincorporation of the products during DNA synthesis in the recipient plants.When ultrasonicated or thermally denatured DNA is applied to the plants it may be degraded and reused for recipient DNA synthesis but it is not recovered in a macromolecular form.The possibility that the intermediate DNA fraction arises by bacterial contamination of the plants can be excluded by several arguments.Autoradiographic studies show that at least part of the radioactivity of the donor DNA taken up by the plants is associated with the cell nucleus.There have been many reports that high-molecularweight DNA can be incorporated by plant protoplasts, intact cells and whole plants. These experiments involved the use of heterologous DNA from various bacteria and phages [l -81. Identification of the foreign DNA occurred mostly by virtue of the different buoyant densities of donor and host DNA. In order to differentiate an exogenous homologous DNA from the recipient DNA by caesium chloride equilibrium centrifugation it is necessary to use donor DNA with an altered buoyant density. This can be obtained by the incorporation of 5-bromodeoxyuridine (BrdUrd) which increases the density of the DNA. If in addition a radioactive label is incorporated, this allows the detection of small amounts of the "heavy" DNA. Uptake of BrdUrd-labelled homologous DNA in a macromolecular form by animal or human cells in culture has been shown [9-121. In this kind of experiment it is necessary to distinguish between the uptake of intact exogenous DNA and the reutilisation of labelled or BrdUrd-containing degradation products of the exogenous DNA for recipient DNA synthesis. This is possible by including unlabelled DNA precursors in the incubation medium. Addition of a large amount of unlabelled thymidine should prevent the utilisation of BrdUrd from degraded BrdUrd-labelled DNA. The incorporation of radioactive degradation products can also be prevented by employing the appropriate competitor.In this report we used a [3H]thymidine or [3H]-adenine-BrdUrd-labelled DNA from Mutthiolu incunu as donor DNA and unlabelled seedlings of Mutthiolu as recipients. The uptake and fate of the DNA was studied by measuring the amount of radioactivity within the plants, by autoradiog...
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