UBC Theses and Dissertations
An examination of in vitro erythropoiesis by utilizing agents that mimic the in vitro activity of erythropoietin Murthy, Sudish C.
The major in vivo hormonal regulator of terminal erythropoiesis is erythropoietin (Ep). This 38,000 dalton acidic glycoprotein has been shown to stimulate the formation of hemoglobinizing erythroblasts. Two in vitro assays designed to measure Ep bioactivity were utilized to determine if other agents could mimic Ep activity in vitro. It was hoped that this approach might yield insights into the mechanism of action of Ep. Several agents have now been identified, and two, dimethyl sulfoxide (DMSO) and sodium orthovanadate had previously been shown (in other systems) to stimulate membrane phosphorylation changes. Accordingly, Ep, DMSO and sodium orthovanadate were assayed with Ƴ-³²P-ATP and plasma membranes purified from Ep-responsive cells to determine if each could induce significant phosphorylation changes as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. It was found that while both sodium orthovanadate and DMSO effected profound phosphorylation alterations, Ep did not elicit any detectable phosphorylation changes. Specifically, vanadate caused a generalized increase in membrane base-stable phosphoproteins, and DMSO reproducibly stimulated the base resistant phosphorylation of a 35 Kd membrane-associated protein. It is reasonable to postulate that the latter phosphorylation event might be responsible for the stimulatory activity of DMSO on terminally differentiating erythroid cells. To understand whether Ep and Ep-mimicking agents were operative on the same target cell population, homogeneous, virally-infected, erythroblasts were cultured in vitro and assayed for ³H-thymidine incorporation in the presence of each agent at various intervals during erythroid cell differentiation. It was found that Ep greatly stimulated very early, as well as differentiated, erythroblasts to proliferate, while four different Ep-mimicking agents could only effect thymidine incorporation into a more mature erythroid population. From this work it is conceivable that Ep-mimicking agents stimulate in vitro erythropoiesis through specific membrane phosphorylation changes and function primarily on late erythroblasts, while the mechanism of action of Ep on primitive and late erythroblasts remains unresolved.
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