losartan-potassium and thioglycerol

The effect of reduced oxygen tension and the role of cellular components known to protect the cell against oxygen toxicity has been studied with respect to erythropoietic colony formation in vitro. Alphathioglycerol can be partially replaced by vitamin E and completely replaced by reduced glutathione (GSH) at physiological concentrations. Incubation of bone marrow and fetal liver early (BFU-E) and late (CFU-E) erythropoietic progenitor cells, in the presence of GSH, in an atmosphere containing 5% oxygen, 5% carbon dioxide and 90% nitrogen, as opposed to air supplemented with 5% carbon dioxide, resulted in an increase in colony numbers and response to erythropoietin (Epo). The number of colonies derived from bone marrow and fetal liver CFU-E increased by 1.2--2.8-fold with a relative Epo sensitivity increase of 3.5--4-fold. Bursts obtained from bone marrow and fetal liver BFU-E increased from 2.6- to 3.8-fold with an increased response to Epo of 2--3-fold. The effects of GSH and low oxygen tension are interpreted as causing a reduction in oxygen toxicity of the cells, thereby increasing the life span in vitro and so increasing the number of cells capable of forming colonies. The heightened response of BFU-E to Epo, analogous to the effect seen for CFU-E, implies that BFU-E may be responsive to physiological Epo concentrations at physiological oxygen tensions.

Topics: Animals; Cell Differentiation; Cell Division; Cells, Cultured; Colony-Forming Units Assay; Dose-Response Relationship, Drug; Erythropoiesis; Erythropoietin; Female; Glutathione; Glycerol; Hematopoietic Stem Cells; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Oxygen Consumption; Vitamin E

Methods for the cultivation of erythroid colonies in vitro are expected to allow selective assay of the earliest committed erythropoietic progenitors in the hemopoietic tissues of man and other species. In the present study, factors affecting erythroid colony formation were examined in methyl cellulose cultures of mouse bone marrow. Efficiency of colony formation observed after 2 days of culture was increased as much as 5-fold (to an average of 325 colonies/10-5 nucleated marrow cells) by the addition of thiol (either beta-mercaptoethanol or alphal-thioglycerol) at a final concentration of 10 minus 4 M. Optimum efficiency required 0.5 erythropoietin units/ml and was influenced by the purity of the preparation. When cultures contained thiol and high doses (3 units/ml) of purified erythropoietin, a second population of erythroid colonies became apparent after 5 days of culture, and increased in size to macroscopic dimensions by the tenth day, when they contained as many as 10-4 cells. Feeding was not required. These colonies, thought to be analogous to the "bursts" reported by Axelrad and coworkers in plasma clot cultures, were observed here at a 6-fold higher frequency (25/10-5 marrow cells) and were linearly related to the number of marrow cells plated, down to limiting numbers of colonies. On the basis of their impressive proliferative potential exhibited in culture, the cells originating these colonies are thought to represent a class of very early erythropoietin-responsive red cell progenitors.

Topics: Animals; Bone Marrow; Bone Marrow Cells; Cell Division; Cells, Cultured; Erythropoietin; Glycerol; Mercaptoethanol; Mice; Mice, Inbred Strains; Sulfhydryl Compounds; Time Factors