Genetic specificity of stress-induced anemia in rats.

There is a genetic difference in rat hemoglobin (Hb) beta-chain structure, with alternate alleles, A and B, at a single locus. This study was designed to find out whether marrow sensitivity due to gamma exposure and experimentally induced anemia in age-matched adult rats is entirely strain specific or is a combination of both strain and Hb genotype. Eight strains of inbred and outbred rats comprising AA and BB types were examined. The data indicate that there is a relationship between marrow sensitivity and Hb genotypes in response to erythropoietic stress caused by three techniques.

Stimulation of newborn-like hemoglobin synthesis in adult rats by recombinant human erythropoietin and suppression by aspirin.

A study was carried out to determine whether recombinant human erythropoietin can induce newborn-like hemoglobin synthesis in adult rats. A fixed dose of recombinant erythropoietin was administered each time intravenously in each rat for altogether 5 weeks. Blood samples drawn at 7-day intervals were analyzed by DEAE-cellulose chromatography. Hematological parameters like red blood cell counts, hematocrit values and reticulocyte counts were evaluated and compared. A significant changing pattern for certain hemoglobin components in red cells of erythropoietin-treated rats was measured compared to their baseline values. However, aspirin (a prostaglandin synthesis inhibitor) intake along with recombinant erythropoietin administration totally abolished the reversion of hemoglobin proportions toward newborn values, but not the increase in hemoglobin synthesis. These data reveal that concurrent prostaglandin synthesis is needed for reversing hemoglobin proportions in adult rats, but not for hemoglobin synthesis per se.

Aspirin blocks 5-azacytidine- and hydroxyurea-induced changes in hemoglobin proportions in adult rats.

The effects of 5-azacytidine and hydroxyurea on their independent ability to change adult hemoglobin proportions toward newborn proportions in adult rats were examined. The results revealed that both the chemotherapeutic agents were capable of switching certain hemoglobin components toward newborn values and required similar time-span to express their actions. However, the switching effect of these drugs was totally lost if aspirin was simultaneously administered into the rats, reflecting the need for concurrent prostaglandin synthesis.

Relationship between plasma PGE2 level and changes in hemoglobin proportions during bleeding-induced anemia in adult rats.

Reversion of hemoglobin proportions toward newborn values is a characteristic change found in blood of acutely bled adult rats. In this study, adult Sprague-Dawley rats were bled over a period of time until they became anemic by hematologic parameters. We measured plasma prostaglandin E2 levels of anemic and control rats using double-antibody technique. A significant increase was recorded in bled plasma, and the value returned to unbled level when anemia was corrected. Acetylsalicylic acid intake during bleeding-induced anemia abolished the process of reverse switching of hemoglobin, as well as inhibited the increase in plasma PGE2 levels. Changes in hemoglobin proportions due to phlebotomy were also blocked when acetylsalicylic acid was replaced by indomethacin. These observations are of significance in understanding, at least in part, the mechanism of reverse hemoglobin switching in adult rats undergoing erythropoietic stress.

Acetylsalicylic acid antagonizes the bleeding-induced changes in hemoglobin proportions in normal adult rats.

Normal adult Sprague-Dawley rats were made anemic by repeated phlebotomy. Ion-exchange chromatography of anemic blood showed newborn like hemoglobin proportions, involving the same six hemoglobin components as is found when newborn and adult blood are compared. However, acetylsalicylic acid intake during anemia failed to demonstrate the changes in hemoglobin proportions, either totally or partially, depending upon the doses. Since acetylsalicylic acid inhibits prostaglandin synthesis, the data suggest that one or more prostaglandins may be involved in the process of reverse switching of hemoglobin in adult rat erythroid cells during erythropoietic stress.

Prostaglandin E2 mediated effects on the synthesis of fetal and adult hemoglobin in blood erythroid bursts.

The effects of prostaglandin E2 (PGE2) in association with erythropoietin on the synthesis of fetal and adult hemoglobin in peripheral blood-derived erythroid burst colonies from normal adults and from patients with sickle cell anemia were investigated. The synthesized hemoglobin at the end of 8, 14 or 18 days in culture was separated by DEAE-cellulose chromatography of 35S-methionine labelled hemoglobin. Quantitative estimation of the synthesized hemoglobin phenotypes, for the three indicated culture periods, showed preferential synthesis of Hb F in addition to an overall increase in hemoglobin synthesis in PGE2 treated colonies. Furthermore, the reactivation of fetal hemoglobin production by PGE2 was more pronounced when the adherent cells were included in the culture dishes. These results indicate that the addition of PGE2 to culture dishes presumably constitutes an environmental change to promote the functional changes seen in the blood erythroid bursts in terms of Hb synthesis and switching.

Comparison of the effects of crude and purified erythropoietin on the synthesis of hemoglobin in rat bone marrow cell cultures.

The stimulatory effects of crude (148 U/mg protein) and purified (15,920 U/mg protein) human urinary erythropoietin (Ep) on heme and globin synthesis of rat bone marrow cells in short term (2-3 day) suspension culture were compared. Freshly isolated, unfractionated marrow cells responded equally well to both Ep preparations. Velocity sedimentation at unit gravity was used to separate the marrow cells into a fast-moving (population I) and a slow-moving (population II) fraction. Of the 2 preparations tested, only crude Ep was found to stimulate heme and globin synthesis of population I cells. Population II cells were unresponsive to both Ep preparations. Preincubation of the rat marrow cells in the absence of Ep for 48 h resulted in the disappearance of most of population II, the remaining cells only responded to the crude Ep preparation. Direct remixing experiments involving various proportions of population I and II cells also showed that addition of the purified Ep preparation could only bring about an increase in hemoglobin synthesis in those cultures which contained a sufficient number of population II cells. Cultures deficient in population II cells to which the crude Ep preparation was added did not show this requirement, suggesting that this preparation contained a component which could substitute for the presence of population II cells. This demonstrates that the action of Ep and of a second factor (or of a cell to cell signal) is required to increase hemoglobin synthesis in short term rat marrow cell cultures.

Rat hemoglobin heterogeneity: postnatal changes in proportions of multiple components and effects of erythropoietin on marrow cell cultures.

Hemolysate from adult and newborn rats was fractionated into nine components (I-IX) using DEAE-cellulose chromatography. A subsequent chromatographic step resolved component IV into two, IVa and Ivb. A comparison of adult and newborn rat hemolysates showed that newborn had higher levels of components I, II, and IV. In the newborn, they accounted for 52% of total hemoglobin eluted, compared to 36% in the adult. Components V, VI, and VIII were less in the newborn than in the adult red cell, at 42% compared to 59%. Minor components III, VII, and IX were unchanged, at 5.8% in newborn and 5.3% in adult rats. Bone marrow cells from adult rats cultured for 46 hours, showed stimulation of eight components under the influence of erythropoietin (components IVa and IVb were not distinguished, and VII was not detected). The extent of stimulation was not consistently higher for major than for minor hemoglobins. These data show that adult rats possess at least ten hemoglobin components, that newborn rats have the same components but in different proportions, and that both major and minor components are stimulated by erythropoietin in bone marrow cell cultures.

Suppression of mouse erythroid colony formation by L1210 leukemia cells.

The effect of L1210 transplantable leukemic cells on in vitro formation of erythroid colonies from CD2F1 mouse bone marrow progenitor cells (CFU-E) was investigated. Clonal cell culture was carried out by a methylcellulose technique. Human urinary erythropoietin served as the stimulator. After 44 hours of incubation aggregates of eight or more erythroid cells were scored as colonies. The number of CFU-E which could be demonstrated in marrow cells from mice that had been injected intravenously 6 days before with 5 x 10(4) L1210 cells was far below that obtained from normal marrow cells. When 1.3 x 10(5) marrow cells from leukemic mice or L1210 ascites cells were cultured with an equal amount of normal cells, the number of CFU-E expressed was reduced by 51% and by 86%, respectively, relative to controls with normal cells only. Neither lethally irradiated L1210 cells (4500 rad) nor L1210 cell conditioned media suppressed erythroid colony formation. It is suggested that in L1210 leukemia erythropoiesis is decreased because of a cell-to-cell inhibitory action of the leukemia cells on CFU-E.