Hypoxia increases erythropoiesis and decreases thrombocytopoiesis in mice: a comparison of two mouse strains

Several previous studies have shown that hypoxia increases erythropoiesis and decreases thrombocytopoiesis in mice. It has been postulated that the thrombocytopenia is caused by stem cell competition between the erythrocytic and megakaryocytic cell lines. In the present work, we compared the effects of severe hypoxia (5.5-6.0% O2) in both male and female C3H and BALB/c mice by measuring their abilities to produce red blood cells and platelets. All mice had significant increases in packed cell volumes and marked decreases in platelet production after hypoxia; however, there were significant differences in the degree of stimulation in the two mouse strains. After 14 days of hypoxia, the percentage of 35S incorporation into platelets, total circulating platelet counts and total circulating platelet masses were lower in C3H mice than in BALB/c mice, but platelet sizes were larger. Also, hypoxia caused greater changes in male mice than in female mice, with male C3H mice showing the greatest increase in packed cell volumes and the lowest platelet counts of all mice tested. The least responses were observed in female BALB/c mice. BALB/c mice had higher P50 (right-shifted O2 dissociation curves) and lower erythrocyte 2,3-diphosphoglycerate values than C3H mice, indicating a lower hemoglobin O2 affinity for BALB/c mice. The results indicate that the effects of hypoxia are not direct upon platelet production, but that the thrombocytopenia is a result of stimulation of erythropoiesis. These data support the stem cell competition hypothesis and illustrate that the degree of the inverse relationship between red blood cells and platelet production of hypoxic mice is dependent, to a large degree, upon the sex and strain of mice that are used.     

Thrombopoietin from human embryonic kidney cells causes increased thrombocytopoiesis in sublethally irradiated mice.

Previous work showed that mice treated with platelet-specific antiserum prior to whole-body irradiation did not suffer the degree or duration of thrombocytopenia as did irradiated control mice. We now report that a partially purified preparation of a thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) mimics the biological effects of platelet-specific antiserum treatment in hematopoietically suppressed mice. Male C3H mice were exposed to 3.0 or 4.5 Gy of 137Cs gamma radiation and injected with a total dose of 4 units (U) of TSF. Human serum albumin (HSA) and rabbit anti-mouse platelet serum-injected mice, along with unirradiated mice, served as controls. Packed cell volumes (PCV), RBC counts, WBC counts, platelet counts, and percentage 35S incorporation into platelets were measured in mice at various days (7-14) following treatment. The results showed that irradiated mice treated with TSF had increased 35S uptake into platelets and higher platelet counts than HSA-treated controls. Also, PCV, RBC counts, and WBC counts of irradiated mice treated with TSF were significantly higher than values for HSA-treated mice. Additional experiments using 40,000 U/mouse of Interleukin-6 (IL-6), 227 U/mouse of granulocyte macrophage-colony stimulating factor (GM-CSF), or a combination of GM-CSF and IL-6 did not show increased platelet counts or 35S incorporation into platelets on Days 10 and 14 when compared to other mice treated with control substances. These results suggest that the radioprotective effects of platelet antibodies reported previously may be due to the release and action of thrombopoietin. These studies also demonstrate that thrombopoietin therapy will modulate the severe thrombocytopenia that occurs in radiation-induced bone marrow suppression.     

Deficient Fe59 and I-125 deoxyuridine uptake by lympho-hemopoietic cell transplants engaged in homograft reactions

Erythropoietic activity of spleen cell grafts was measured (Fe⁵⁹ uptake) in X-irradiated recipient mice under conditions in which these grafts were engaged in homograft reactions against allogeneic target cells or in graft-versus-host reactions. Such Fe⁵⁹ incorporation was greatly reduced at 7 to 10 days after graft implantation relative to that of control grafts. This reduced erythropoiesis did not occur when the spleen cell graft was immunologically incompetent. Transplantation of bone marrow-lymph node cell mixtures also resulted in a relative decline in Fe⁵⁹ uptake, but only when minimal numbers (10⁵ to 10⁶) of marrow cells were injected. The incorporation of I¹²⁵ UdR in the spleen of irradiated recipients was used to assess cellular proliferation. Incorporation of this label was reduced when measured 7–10 days after implantation of the lympho-hemopoietic cell graft, but reached a peak at five days—the latter indicating stimulated lymphopoiesis. These data are consistent with the concept of depletion of a pluripotent stem cell pool (limited in size under these experimental conditions) due to excessive and concurrent functional demands for erythropoiesis and lymphopoiesis. An alternative explanation would involve cytotoxic effects on hemopoietic elements present in the milieu of the immunologic reaction.     

Thyroxine suppresses thrombocytopoiesis and stimulates erythropoiesis in mice.

Thyroxine has been shown in vitro to stimulate erythropoiesis by two mechanisms: a direct, beta 2-adrenergic receptor-mediated stimulation of red cell precursors, and an indirect, erythropoietin-mediated mechanism. Clinical reports have suggested that excess thyroxine also exerts depressive effects on thrombocytopoiesis, but the most sensitive methods of assessing platelet production, i.e., percentage of 35S incorporation into platelets and determination of megakaryocyte size and number, are not appropriate for analysis of platelet production in human patients. The purpose of this study was to use a mouse model to investigate the effects of the hyperthyroid state on erythropoiesis and thrombocytopoiesis, and to assess in vivo the two mechanisms by which thyroxine has been described to stimulate erythropoiesis in vitro. We found that thyroxine administration significantly depressed platelet production and stimulated erythropoiesis in mice. Both the D- and L-isomers of thyroxine in appropriate doses produced this depression of thrombocytopoiesis, and the effect was dose dependent for both isomers. Daily administration of thyroxine:increased blood volume; decreased the peripheral platelet count, total circulating platelet count and mass, percentage of 35S incorporation into platelets, and megakaryocyte number and size; and concurrently increased indices of red cell production (packed cell volume, red blood cell count, total circulating red blood cell count and mass, and reticulocyte count). Additionally, propranolol, a nonspecific beta-blocker, partially reversed the suppression of platelet production by L-thyroxine, lending credence to the assertion that the direct, beta 2-adrenergic receptor-mediated stimulation of the erythroid cell line by thyroxine reported to exist in vitro may also be important in vivo.     

Effects of hypoxia on the small acetylcholinesterase-positive megakaryocyte precursor in bone marrow of mice

The number of small acetylcholinesterase-positive (SAChE+) cells in the marrow of hypoxic mice was measured. Mice were exposed to 6-7% O2 levels by enclosure in cages covered with dimethyl-silicone rubber membranes for 1-14 days. The mice showed a linear increase in packed cell volumes with time in the hypoxic atmosphere, but platelet counts showed a characteristic biphasic response, i.e., increased platelet counts were observed after 1-3 days of hypoxia, and significantly (P less than 0.05-P less than 0.0005) decreased platelet counts were observed thereafter (6-14 days). The total number of megakaryocytes in the marrow of hypoxic mice decreased significantly (P less than 0.005) with time. In agreement with the data on platelet counts, hypoxia caused the total number of SAChE+ cells in the marrow of mice to be biphasic. At Day 2 there was a significant increase (P less than 0.05) in the total number of SAChE+ cells/mm3 of bone marrow; however, by days 10-14 the numbers had decreased markedly (P less than 0.005). These data indicate that hypoxia decreases platelet production by action on a precursor cell to the SAChE+ cell. The hypoxia-induced thrombocytopenia is probably caused by stem-cell competition between the erythrocytic and megakaryocytic cell lines.     

Induction of metallothionein by zinc in lethal milk mutant mice

Adult lethal milk (lm/lm) mutant mice display increased induction of hepatic metallothionein synthesis compared to wild-type mice following the subcutaneous injection of 40 µmol ZnCl₂/kg mouse. At this zinc dose the rate of incorporation of |³⁵S| cysteine into hepatic metallothionein in adult (100-to 230-day-old) lm/lm mice was approximately 2.4-fold greater than the rate of incorporation of isotope in wild-type animals. At a higher zinc dose (160 µmol ZnCl₂/kg) the incorporation of |³⁵S| cysteine into hepatic metallothionein was similar in lm/lm and wild-type mice. The altered dose-response to zinc administration was not due to a change in hepatic zinc, copper, or manganese levels, to a difference in ⁶⁵Zn uptake, or to an alteration in ⁶⁵Zn bound to differential centrifugation fractions of adult lm/lm liver. ⁶⁵Zn bound to hepatic metallothionein was, however, increased in aging lm/lm mice with symptomatic skin lesions.     

Effect of zinc on the biosynthesis of indole glucosinolates glucobrassicin and neoglucobrassicin in etiolated seedlings of rape (Brassica napus var.arvensis (Lam.) Thell)

The effect of Zn²⁺ ions (in the form of ZnCl₂) in the ceoncentration range 10^?3 to 10^?6 M on the content and biosynthesis of indole glucosinolates glucobrassicin and neoglucobrassicin has been studied on etiolated seedlings of rape (Brassica napus var.arvensis (Lam.) Thell). In the “long-term” experiment zine ions influenced the seedlings during eight days of germination, whereas in the “short-term” experiment zinc ions acted only 72 h on seven days old intact seedlings. The biosynthesis of indole glucosinolates has been followed by the incorporation of³⁵S from Na₂ ³⁵SO₄ into both glucosinolates in experiments with, hypocotyl segments of the rape seedlings. Zinc ions at chronic “long-term” application increased the glucobrassicin and neoglucobrassicin level in the seedlings. The neoglucobrassicin content especially was increased. A “short-term” application of zinc ions increased the level of both glucosinolates at higher and lower concentrations, whereas medium concentrations (10^?4 and 10^?5 M) lowered their level. Zn²⁺ ions lowered absorption of³⁵SO₄ ^?2 ions by hypocotyl segments and simultaneously lowered the incorporation of³⁵S into glucobrassicin. On the contrary, the incorporation of³⁵S into neoglucobrassicin and proteins was stimulated. Zinc ions do exhibit a specific effect on neoglucobrassicin biosynthesis, on membrane permeability as against sulphate ions and on the incorporation of sulphur into proteins.     

The effect of temperature on the uptake and loss of anions by seedling roots ofZea mays L

Intact maize seedlings were examined for the uptake and leakage of labelled sulphate and phosphate anions affected by temperature. Control plants, grown at 25 °C were compared from the aspects of uptake capacity and leakage with plants incubated in nutrient solutions cooled to 15 °C and 5 °C, respectively. Short time intervals as well as 1–7 d exposure to cooling were used. Already after 1 h exposure at 5 °C and 5 h cooling at 15 °C and at 5 °C, considerable changes were manifested in anion uptake and leakage. The uptake of³²P declined more than that of³⁵S. So, after a 30 min uptake interval the uptake of³⁵S decreased at 15 °C to 49.84% and at 5 °C to 6.05% comparing with the uptake at 25 °C, while the uptake of³²P declined to 28.64% at 15 °C and to 4.45% at 5 °C. The leakage of both anions was the highest at 25 °C in absolute rates, but relatively most of the uptaken³⁵S and³²P was released at 5 °C. Longer exposure to a chilling temperature of 5 °C (1–7 days) resulted in two patterns of sulphate and phosphate uptake.     

The effect of high altitude on platelet counts, thrombopoietin and erythropoietin levels in young Bolivian airmen visiting the Andes

Recognition of thrombosis as a complication of exposure to high altitude has stimulated interest in rheological changes resulting from hypobaric hypoxia. Previous studies of platelet counts at high altitude have yielded conflicting results and have not been studied in conjunction with potential mediating cytokines. We studied the effects of high-altitude exposure on platelet numbers, thrombopoietin (tpo) and erythropoietin (epo) levels in man. A group of 28 volunteers from the Bolivian Airforce stationed at Santa Cruz (600 m altitude) were studied 48 h and 1 week after their ascent to La Paz (3600 m). In addition 105 volunteers based at Santa Cruz for at least 1 year were compared with 175 age- and sex-matched residents at El Alto (4200 m). Platelet counts were measured immediately after sampling and serum samples assayed for tpo and epo. In the ascending group, mean platelet counts were 251×10⁹, 367×10⁹ and 398×10⁹/l at 600 m and following 48 h and 1 week at 3600 m respectively. Mean tpo levels were 132.5, 76 and 92 pg/ml with epo values of 2.98, 11.6 and 7.9 mIU/ml respectively. In the resident populations mean platelet counts were 271×10⁹/l in the low- and 471×10⁹/l in the high-altitude groups. Mean tpo and epo levels measured 69.3 pg/ml and 4.5 mIU/ml respectively at 600 m and 58.5 pg/ml and 5.1 mIU/ml at 4200 m. In conclusion we have demonstrated a significant and sustained elevation in platelet numbers within 48 h of ascent to high altitude. Our findings do not support a role for tpo as a mediator of the increased platelet count. However, these data do not discount epo as a potential candidate. Received: 28 December 1998 / Revised: 13 May 1999 / Accepted: 26 May 1999     

Magnesium-Dependent stimulation of protein synthesis by the insulin mimic,pervanadate

The insulin mimic, peroxide of vanadate (pervanadate), stimulated ³⁵S-methionine incorporation into Xenopus oocyte protein in a Mg²⁺-dependent manner. Reducing the extracellular Mg²⁺ concentration from 1.0 to 0.1 mM decreased the pervanadate-stimulated component of incorporation by 35%; with 0.01 mM Mg²⁺ or lower, the pervanadate-stimulated component was abolished. In addition, reducing extracellular Mg²⁺ to 0.01 mM inhibited about 50% of the insulinstimulated component of methionine incorporation. Mg²⁺ depletion had no effects on incorporation in controls or when protein synthesis was stimulated by Zn²⁺ or bovine growth hormone. Thus, not all substances that stimulated protein synthesis showed a dependence on extracellular Mg²⁺. Reducing extracellular Ca²⁺ had no effects on methionine incorporation in control cells or in cells stimulated by pervanadate or insulin. When oocytes maintained in a paraffin oil medium were brought into contact with a 0.5 m?I droplet of buffer containing the Mg²⁺ indicator dye, mag-fura-2, and pervanadate, apparent droplet Mg²⁺ decreased rapidly, indicating net uptake by the cells. Insulin also caused a net uptake of Mg²⁺. In contrast, apparent extracellular Mg²⁺ was constant when cells were in contact with droplets containing no effectors. Together, these data indicate that extracellular Mg²⁺, but not Ca²⁺, is involved in the stimulation of protein synthesis by pervanadate, and to a lesser extent by insulin. Pervanadate appears to induce a net uptake of Mg²⁺, and this change in membrane transport may be an early event in signalling the increase in translation. © 1995 Wiley-Liss, Inc.     

Transformations and plant uptake of urine-sulphate in urine-affected areas of pasture soil

The fate of sheep urine sulphate in the soil and its plant uptake was monitored using ³⁵S-labelled sulphate-S in undisturbed pasture microplots in two glasshouse experiments. The extent of macropore flow of simulated urine immediately following a sheep urination was also investigated at 5 pasture sites in the field. Immediately following urination to pasture microplots in the glasshouse, the amounts of urinederived ³⁵S recovered in the 0–2.5, 2.5–7.5, 7.5–15 and 15–30 cm soil layers were 38, 28, 18 and 9%, respectively. In the field study on 5 contrasting soils, a similar pattern was found with 55–70, 20–35 and 13–20% of simulated urine being recovered in the 0–5, 5–10 and 10–15 cm soil layers, respectively. There was insignificant loss below 15 cm. If urine had moved via simple displacement in these soils the wetting front would have reached only 2.0–2.5 cm in depth suggesting that significant downward movement of urine via macropore flow occurs after urination. In a 15-day period following urine application to a pasture soil there was a rapid rate of incorporation of ³⁵S into organic forms, while between 15 and 64 days the rate of incorporation declined. After 7 days, 27% of added ³⁵S had been incorporated into organic forms with 19% being C-bonded S and 8% Hl-reducible S. This rapid incorporation was attributed to the large and active microbial biomass present in the rhizosphere. Since urine application depressed pasture growth, due to ‘urine burn’, less than 10% of applied ³⁵S was absorbed by pasture plants over a 64-day period. A second experiment using microplots of contrasting soil types, confirmed that the majority of the ³⁵S incorporated into the organic form was present as C-bonded S. Results showed that of the ³⁵S remaining in the 0–2.5 cm layer 35 days after application, 20–40% was present as sulphate, 10–20% as Hl-reducible S and 50–60% as C-bonded S. Plant uptake of S accounted for only 7–12% of applied ³⁵S over the 35-day period.     

Effect of copper on the content and the biosynthesis of indole glucosinolates glucobrassicin and neoglucobrassicin in etiolated rape seedlings (Brassica napus var.arvensis (Lam.) Thell.)

The influence of Cu²⁺ ions (in the form of CuCl₂) in the concentration range 10^?3 to 10^?6 M on the content and biosynthesis of indole glucosinolates glucobrassicin and neoglucobrassicin has been studied on etiolated seedlings of rape (Brassica napus var.arvensis (Lam.) Thell.). Ions Cu²⁺ acted on the seedlings either chronically from the beginning of the germination or acutely, during 3 to 72 h, on seven days old seedlings. The biosynthesis of both glucosinolates was followed by the incorporation of³⁵S from Na₂ ³⁵SO₄ into them in hypocotyl segments from seven days old intact etiolated seedlings. After the entry of small amounts of Cu²⁺ ions into the plants, stimulation of the glucosinolates formation occurs, as was found after three h action of Cu²⁺ ions. After the entry of a greater amount of Cu²⁺ ions into the plant, harmful effects appear, as was found after chronic two days action or after 24 and 48 hours acute action of Cu²⁺ ions. Later further stimulation of glucosinolate formation occurs, probably due to enhanced metabolism during reparation processes, as was manifested after chronic action of Cu²⁺ ions lasting four and eight days. The optimal effect of copper was found mainly in the concentration range 5×10^?4 M to 10^?5 M. Ions Cu²⁺ in higher concentration increased the uptake of sulphate ions by hypocotyl segments, and in lower concentrations increased the incorporation of³⁵S from³⁵SO₄ ^2? into the proteins.     

Effect of ESF preparations on different types of erythroblasts

A new method of quantitative ¹⁴C-autoradiography was applied for evaluating possible effects of erythropoietin (ESF) on the DNA synthesis rate of differentiated erythroid murine bone marrow cells identified as proerythroblasts, basophilic and polychromatic erythroblasts. Eosinophilic myelocytes were used as a control cell population. ESF was prepared from the urine of a patient with chronic aplastic anemia; an inactive urinary preparation served as control. The potency of the preparations was estimated by the ⁵⁹Fe-incorporation assay. The materials to be tested were injected into polycythemic mice 4, 8 and 16 hours before in vitro short-term incubation of the bone marrow cells with ¹⁴C-thymidine and Methotrexate. Animals without test material were taken as additional controls. Autoradiographic grains were counted by an incident light microscope photometer. Eight hours after injection of ESF a significant increase in the mean ¹⁴C-thymidine incorporation was found in all three erythroid cell types when compared either with the inactive control preparation (excess incorporation 30–40%) or with the untreated control animals (excess incorporation 10–20%). It could be shown that the increase is due to an immediate action of ESF on already differentiated cells and cannot –- at least not solely –- be attributed to its action on hemopoietic stem cells. The control preparation which was inactive in terms of ⁵⁹Fe incorporation exerted a slight inhibition of DNA synthesis rate in all erythroid cells as well as in cells not committed to erythroid differentiation.     

14 C-acetate incorporation and nucleic acid synthesis in human lymphocytes stimulated by PHA and tuberculin in vitro

Studies on the timing of incorporation of labeled acetate in relationship to other cellular events in phytohemagglutinin (PHA)-treated lymphocytes have suggested that acetylation of nuclear histones may constitute an important regulatory mechanism for gene activation. In the present investigation, it was shown that PHA stimulation of lymphocytes from a tuberculin-positive patient caused an early increased incorporation of ¹⁴C-acetate prior to RNA and DNA synthesis. Lymphocytes from the same patient, however, repeatedly showed no increased incorporation of ¹⁴C-acetate following exposure to the sensitizing antigen, tuberculin (PPD), even though RNA and DNA synthesis were markedly stimulated. These results suggest that regulatory mechanisms of DNA template activity other than acetylation may be operative in sensitized lymphocytes responding to specific antigen. One possible explanation for the differences in ¹⁴C-acetate incorporation is that the increased uptake of acetate exhibited by PHA-treated cells is an effect related to nonspecific membrane changes caused by the PHA. If this is the case, then template regulation in PHA and antigen-stimulated lymphocytes may be achieved via similar but yet to be defined mechanisms.     

Some effects of aluminium on rat brain protein synthesis

1. Infant rats and rabbits received intraperitonal aluminium (Al) chloride (5, 10 or 20 mg Al/kg body weight) every third day from one to four weeks of age.2. When the polysomal fraction was tested in a protein synthesizing system, a significant increase in the incorporation of [¹⁴C] leucine, [¹⁴C] phenylalanine, or [³⁵S] methionine into proteins in vitro was observed at the higher doses in rats but not rabbits.3. The incorporation of [³⁵S]methionine into brain ferritin was measured using polysomal mRNA or mRNA “stored” in the ribonucleoprotein (RNP) particle fraction.4. The results suggest that Al exposure causes the mobilization of ferritin mRNA from the latter fraction to the polysomal fraction for increased ferritin synthesis.     

Degradation of 35S-labeled metallothionein in the liver and the kidney of Brindled mice: Model for Menkes' disease

An amino acid analysis of the renal copper-binding protein of heterozygous Brindled mice indicated that the protein labeled with L-[³⁵S]cystine was metallothionein.The metabolism of ³⁵S-labeled hepatic and renal metallothionein of adult normal (Mo^+/+) and heterozygous (Mo^br/+) Brindled mice was investigated without prior induction with metals. After incorporation of L-[³⁵S] cysteine into hepatic and renal metallothionein, ³⁵S-labeled metallothionein is normally degraded with two half-lives (liver: 11.6 ± 1.3 hours and 3.1 ± 0.3 days; kidney: 8.22 ± 0.08 hours and 3.5 ± 1.2 days). However, ³⁵S-labeled renal metallothionein of the heterozygous Brindled mice is exclusively degraded with a half-life of 3.1 ± 0.2 days.The results imply that the mutation in Brindled mice causes an impaired renal reabsorption of copper (transport of copper from the tubular cells into the blood circulation).     

Synthesis and radiobiological applications of [35S]l-homocysteine thiolactone

[³⁵S]l-Homocysteine thiolactone ([³⁵S]l-HCTL) was synthesized and its biodistribution evaluated as a potential brain radioprotective agent and as a tissue hypoxia marker. Drug uptake in mouse brain exceeded that in s.c. tumor 3 h post injection only. Multiple indicator dilution experiments in the rabbit heart indicate that membrane permeability of [³⁵S]l-HCTL does not limit its usefulness as a hypoxia marker. In addition, a positive correlation was observed between regional coronary blood flow and myocardial content of [³⁵S]adenosylhomocysteine formed from [³⁵S]homocysteine and adenosine.     

Inflammation neither increases hepatic hepcidin nor affects intestinal 59Fe-absorption in two murine models of bowel inflammation,hemizygous TNFΔARE/+ and homozygous IL-10−/− mice

Hepcidin-synthesis was reported to be stimulated by inflammation. In contrast, hepcidin synthesis was inhibited by TNFα and serum hepcidin was low. To elucidate these contradictions, we compare data on hepcidin expression, on iron absorption and homoeostasis and markers of inflammation between two murine models of intestinal inflammation and corresponding wild-types as determined by standard methods.In TNF^ΔARE/+ and IL-10^−/−-mice hepatic hepcidin expression and protein content was significantly lower than in corresponding wild-types. However, ⁵⁹Fe whole-body retention showed no difference between knock-outs and corresponding wild-types 7d after gavage, in neither strain. Compared to wild-types, body weight, hepatic non-haem iron content, hemoglobin and hematocrit were significantly decreased in TNF^ΔARE/+ mice, while erythropoiesis increased. These differences were not seen in IL-10^−/− mice. Duodenal IL-6 and TNFα content increased significantly in TNF^ΔARE/+ mice, while ferritin-H decreased along with hepatic hepcidin expression, ferritin L, and non-haem iron. In IL-10^−/− mice, these changes were less marked or missing for non-haem iron. Duodenal ferritin-L and ferroportin increased significantly, while HFE decreased.Our results corroborate the conflicting combination of low hepcidin with inflammation and without increased intestinal iron absorption. Speculating on underlying mechanism, decreased hepcidin may result from stimulated erythropoiesis. Unaltered intestinal iron-absorption may compromise between the stimulation by increased erythropoiesis and inhibition by local and systemic inflammation. The findings suggest intense interaction between counterproductive mechanisms and ask for further research.