Effect of acute hypoxia on the renin and erythropoietin activities of the blood serum in rats

A comparative study of renin and erythropoietin content in the blood serum of rats with "endocrine" kidneys and changes in their activity following the action of specific erythropoietic stimulus (4-hour hypoxia) has been conducted. The presence of "endocrine" kidneys increased renin and erythropoietin activity in such animals. Acute hypoxia produced further increase in erythropoietin titre in the blood serum, with renin remaining at the same level. Possible differences in the mechanisms of renin and erythropoietin biogenesis in the kidneys are considered.     

Erythropoietic function of the kidneys under conditions of abnormally high oxygen pressure]

Three to five hours after 5-hour exposure of rabbits to high oxygen pressure (2 ata) the erythropoietin proved to disappear from both the arterial and the venous blood plasma of the kidneys. At the same time the blood plasma from the renal vein began to suppress the mitotic activity of erythroblastic cells in the bone marrow culture. These data testify to the fact that under hyperbaric hyperoxia the kidneys secreted the inhibitor of erythropoiesis. No erythropoiesis inhibitor was revealed 24 hours after the hyperoxia.     

On the mechanism of erythropoietin-induced differentiation. XII. A cytoplasmic protein mediating induced nuclear RNA synthesis

Erythropoietin, the primary inducer of red blood cell differentiation, has no effect on RNA synthesis by isolated bone marrow nuclei. A cytoplasmic fraction from marrow cells exposed to erythropoietin does, however, stimulate RNA synthesis by such nuclei. This marrow cell cytoplasmic factor (MCF) also stimulates RNA synthesis by liver and kidney nuclei, whereas erythropoietin has no effect on intact kidney or lung cells. MCF appears rapidly in cells after addition of erythropoietin, and its formation does not require protein synthesis. MCF is inactivated by trypsin, but not by ribonuclease. The data suggest that erythropoietin acts on the responsive cells to generate a cytoplasmic protein that mediates the effect of the hormone on nuclear RNA synthesis.     

Impaired erythropoietin synthesis in chronic kidney disease is caused by alterations in extracellular matrix composition

Renal fibrosis and anaemia are two of the most relevant events in chronic kidney disease. Fibrosis is characterized by the accumulation of extracellular matrix proteins in the glomeruli and tubular interstitium. Anaemia is the consequence of a decrease in erythropoietin production in fibrotic kidneys. This work analyses the possibility that the accumulation of abnormal collagens in kidney interstitium could be one of the mechanisms responsible for erythropoietin decreased synthesis. In renal interstitial fibroblast grown on collagen I, erythropoietin mRNA expression and HIF‐2α protein decreased, whereas focal adhesion kinase protein (FAK) phosphorylation and proteasome activity increased, compared to cells grown on collagen IV. Proteasome inhibition or FAK inactivation in cells plated on collagen I restored erythropoietin and HIF‐2α expression. FAK inhibition also decreased the collagen I‐dependent proteasome activation. In a model of tubulointerstitial fibrosis induced by unilateral ureteral obstruction in mice, increased collagen I protein content and an almost complete disappearance of erythropoietin mRNA expression were observed in the ureteral ligated kidney with respect to the contralateral control. Interestingly, erythropoietin synthesis was recovered in obstructed mice treated with proteasome inhibitor. These data suggest that reduced kidney erythropoietin synthesis could be caused by the accumulation of abnormal extracellular matrix proteins.     

Evidence for an erythropoietin receptor protein on rat bone marrow cells

Rat bone marrow cells, $\text{in vitro}$, respond to erythropoietin by increased RNA synthesis even in the absence of protein synthesis. Trypsin treated cells lose their ability to respond to the hormone if protein synthesis is inhibited, but retain responsiveness if protein synthesis is permitted during the incubation. The data suggest that a protein receptor on the external surface of the responsive cells is required for the action of erythropoietin on marrow cells.     

Appearance of host-specific nucleolar proteins in intranuclear "dense bodies" following herpes simplex infection

Nucleolar modifications induced by herpes simplex virus type 1 (HSV1) infection were studied at the ultrastructural level with special attention to the fate of a family of proteins serologically related to the nucleolar 100 kDa protein. Immunocytochemical techniques revealed that antigenic sites related to these proteins were associated with nucleoli in a pattern similar to that observed with non-infected cells. In addition, the "dense bodies" induced by HSV infection were heavily decorated by antibodies to the 100 kDa protein. Neither DNA nor RNA was detectable in the latter by cytochemical techniques. Therefore, it appears that "dense bodies" are exclusively proteinaceous and contain at least one host protein implicated in ribosomal RNA synthesis. An accumulation of 100 kDa protein in extra-nucleolar structures might account for previously reported defects in ribosomal RNA expression during HSV infection.     

alpha-Tocopherol transfer protein expression in rat liver exposed to hyperoxia

-Tochopherol transfer protein ( TTP), a 32 kDa protein exclusively expressed in liver cytosol, has a high binding affinity for -tochopherol. The factors that regulate the expression of hepatic TTP are not clearly understood. In this study, we investigated whether or not exposure to hyperoxia (95% O 2 for 48 h) could alter the expression of hepatic TTP. We also examined the association between the expression of antioxidant enzymes (hepatic glutathione peroxidase (GPX) and Mn-superoxide dismutase (Mn-SOD)) and the expression of hepatic TTP. The levels of thiobarbituric acid-reactive substances (TBARS) in both plasma and liver were significantly higher after rats were exposed to hyperoxia for 48 h when compared with the levels in control rats. Northern blotting showed a decrease in the expression of TTP messenger RNA (mRNA) after hyperoxia, although the TTP protein level remained constant. Expression of Mn-SOD mRNA and protein, as well as the expression of GPX mRNA, were stable after hyperoxia. These findings indicate that mRNA for hepatic TTP, rather than Mn-SOD or GPX, may be highly responsive to oxidative stress.     

Serum erythropoietin levels in healthy humans after a short period of normobaric and hyperbaric oxygen breathing: the "normobaric oxygen paradox".

Renal (peritubular) tissue hypoxia is a well-known physiological trigger for erythropoietin (EPO) production. We investigated the effect of rebound relative hypoxia after hyperoxia obtained under normo- and hyperbaric oxygen breathing conditions. A group of 16 healthy volunteers were investigated before and after a period of breathing 100% normobaric oxygen for 2 h and a period of breathing 100% oxygen at 2.5 ATA for 90 min (hyperbaric oxygen). Serum EPO concentration was measured using a radioimmunoassay at various time points during 24-36 h. A 60% increase (P < 0.001) in serum EPO was observed 36 h after normobaric oxygen. In contrast, a 53% decrease in serum EPO was observed at 24 h after hyperbaric oxygen. Those changes were not related to the circadian rhythm of serum EPO of the subjects. These results indicate that a sudden and sustained decrease in tissue oxygen tension, even above hypoxia thresholds (e.g., after a period of normobaric oxygen breathing), may act as a trigger for EPO serum level. This EPO trigger, the "normobaric oxygen paradox," does not appear to be present after hyperbaric oxygen breathing.     

"Chromatomics" the analysis of the chromatome

Chromatin is a highly complex mixture of proteins and DNA that is involved in the regulation and coordination of gene expression within the eukaryotic nucleus. Changes in chromatin structure can convey heritable changes of gene activity in response to external stimuli without altering the primary DNA sequence. This epigenetic inheritance of particular traits very likely plays a major role during evolutionary processes. It is however, still ill-defined how this non DNA-mediated inheritance is accomplished at a molecular level. The advent of new methods to systematically study genome-wide changes in chromatin condensation, DNA methylation levels, RNA synthesis and the association of specific proteins or protein modifications now allows a thorough investigation of changes in chromatin structure and function in response to environmental alterations. We would like to review some of these global approaches and to introduce the term "chromatomics" for the systematic analysis of the DNA, RNA and protein content of the genetic material in the eukaryotic nucleus.     

On the mechanism of erythropoietin-induced differentiation : XV. Induced transcription restricted by cytosine arabinoside

We have examined the role of DNA synthesis in the induced differentiation of erythropoietin-responsive cells (ERC) by using cultured marrow cells from plethoric rats. In such marrow cell populations there are minimal numbers of differentiated erythroid cells permitting the study of erythropoietin action on the non-differentiated primitive ERC. Cytosine arabinoside (10⁻⁴ M) and 5-fluorodeoxyuridine (10⁻⁷ M) were used for inhibition of DNA synthesis. The data indicate that DNA synthesis is not required for the early steps in the initiation of RNA synthesis or hemoglobin synthesis by erythropoietin. The evidence suggests, however, that ERC may be sensitive to erythropoietin only in a cell cycle phase after S. This period, presumably in G2, is approx. 85 min long. The full response to erythropoietin, therefore, requires DNA synthesis both to replenish the G2 compartment and to permit amplification divisions of induced cells.     

Nitric-oxide Synthase-2 Linkage to Focal Adhesion Kinase in Neutrophils Influences Enzyme Activity and β2 Integrin Function

This investigation was to elucidate the basis for augmentation of nitric-oxide synthesis in neutrophils exposed to hyperbaric oxygen. Hyperoxia increases synthesis of reactive species leading to S-nitrosylation of β-actin, which causes temporary inhibition of β₂ integrin adherence. Impaired β₂ integrin function and actin S-nitrosylation do not occur in neutrophils from mice lacking type-2 nitric-oxide synthase (iNOS) or when incubated with 1400W, an iNOS inhibitor. Similarly, effects of hyperoxia were abrogated in cells depleted of focal adhesion kinase (FAK) by treatment with small inhibitory RNA and those exposed to a specific FAK inhibitor concurrent with hyperoxia. Nitric oxide production doubles within 10 min exposure to hyperoxia but declines to approximately half-maximum production over an additional 10 min. Elevated nitric oxide production did not occur after FAK depletion or inhibition, or when filamentous actin formation was inhibited by cytochalasin D. Intracellular content of iNOS triples over the course of a 45-min exposure to hyperoxia and iNOS dimers increase in a commensurate fashion. Confocal microscopy and immunoprecipitation demonstrated that co-localization/linkage of FAK, iNOS, and filamentous actin increased within 15 min exposure to hyperoxia but then decreased below the control level. Using isolated enzymes in ex vivo preparations an association between iNOS and filamentous actin mediated by FAK could be demonstrated and complex formation was impeded when actin was S-nitrosylated. We conclude that iNOS activity is increased by an FAK-mediated association with actin filaments but peak nitric oxide production is transient due to actin S-nitrosylation during exposure to hyperoxia.     

Some studies of erythroid differentiationin vitro

Summary When rat marrow cells are pre-incubated for periods of up to 20 days they retain the capacity to respond to exogenous erythropoietin. This is true whether the response is measured in terms of increased rates of RNA synthesis, protein synthesis, iron-uptake, or hemoglobin synthesis. The patterns of response to erythropoietin by pre-incubated cells are complex and involve secondary waves of increased rates of synthesis that are not yet understood. Life Insurance Medical Research Fund Fellow. Operated by the University of Chicago for the United States Atomic Energy Commission.     

Effects of dexamethasone on the levels of plasma corticosteroid binding globulin in rats and monkeys.

Rat marrow cells were preincubated for 45 hours with 5.5 × 10^?4M sodium hexachloroiridate. This treatment abolished DNA synthesis whilst improving cell survival over that of controls. The synthesis of RNA, protein and glycoprotein continued and could be further increased by the addition of erythropoietin for up to 44 more hours. Heme synthesis also continued in the absence of DNA synthesis but could not be stimulated by erythropoietin.     

Assessment of the erythropoietin-producing function of the kidney and liver under controlled perfusion

The balance of erythropoietin production by the dog kidney and liver was studied during controlled normoxic perfusion. The hormone production was stimulated by acute posthemorrhagic anemia (bloodletting of 25% total blood volume) combined with subcutaneous injection of cobaltous chloride (250 microM/kg body weight). The increase in erythropoietin level was revealed in posthypoxic animal perfusate after 6 hours of perfusion. The amount of hepatic erythropoietin was shown to be 2.5 times higher than that excreted by kidneys.     

Isolation and characterization of poly(A)-containing polyoma "early" and "late" messenger RNAs.

During late lytic infection of mouse kidney cell cultures polyoma 16S and 19S (late 19S RNA) were isolated by oligo(dT)-cellulose chromatography. Approximately 60-80% of total cytoplasmic polyoma RNA contained tracts of poly(A) which were retained by oligo(dT)-cellulose. Early in lytic infection when viral DNA synthesis and the production of capsid protein are blocked by the addition of 5-fluorodeoxyuridine, approximately 100% of polyoma "early" 19S RNA was quantitatively retained by oligo(dT)-cellulose indicating the presence of poly(A) tracts on most 19S mRNA molecules. In addition, 2 classes polyoma RNA, synthesized after the onset of cellular RNA synthesis under conditions where DNA synthesis is inhibited with 5-fluorodeoxyuridine, were found to contain tracts of poly(A). These species sedimenting at 16S and 19S in aqueous sucrose density gradients were also quantitatively retained by oligo (dT)-cellulose.     

Protein synthesis in hyperoxic endothelial cells: evidence for translational defect

To study the effects of hyperoxia on protein synthesis in primary cultures of porcine aortic endothelial cells, we exposed confluent cells to different O2 concentrations for various durations. Exposure to 95% O2 for 5 days resulted in a 71% inhibition of [3H]phenylalanine incorporation into total proteins. When compared with control cells, we observed no changes in 1) the pool size of free cytoplasmic phenylalanine and of phenylalanine attached to transfer RNA (tRNA), 2) the rate of protein degradation, and 3) the rate of charging of tRNA with phenylalanine. We found that under hyperoxic conditions 1) the incorporation of [3H]-uridine into total and polyadenylated RNA was increased, 2) the efficiency of extracted messenger RNA to direct protein synthesis in a reticulocyte lysate was maintained, 3) the proportion of polymeric to monomeric ribosomes was slightly increased, and 4) the rate of elongation, as measured by the ribosomal transit time, was decreased. Thus the reduction in protein synthesis in hyperoxic cells appears to result primarily from defects at the translational level in polypeptide chain elongation.     

Dependence between the rate of RNA synthesis and the CTP-synthetase activity of different rat and chicken tissue

The rate of total RNA synthesis determined by the method of Bukher and Sheffield in different tissues of rats and chickens decreases as follows: kidneys greater than liver greater than duodenum mucosa. The activity of CTP-synthetase (EC 6, 3, 4, 2) determined in vitro and in vivo by the method suggested correlates with the rate of total RNA synthesis in the kidneys and liver tissues of rats and chickens. It is shown that 34-35% of CTP synthetized de novo in the chicken tissues and 31-34% in the rat tissues are used for RNA synthesis. At the same time 75-90% of CTP incorporating into RNA is formed by salvage pathway.