The regulation of porphobilinogen oxygenase and porphobilinogen deaminase activities in rat bone marrow under conditions of erythropoietic stress.

Porphorbilinogen oxygenase (EC 4.2.1.24) was associated with the microsomal fraction of bone marrow in normal rats and in rats submitted to erythropoietic stress, while porphobilinogen deaminase (EC 4.3.1.8) of the same origin was present in the cytosol. An NADPH-dependent electron-donor system for the oxygenase was also present in the microsomes of the bone marrow. Under conditions of erythropoietic stress caused by hypoxia, the activities of both enzymes were found to be inversely correlated. While the oxygenase showed minimum activity between the 4th and 8th day of hypoxia, porphobilinogen deaminase reached its maximum activity during this period. After the 8th day of hypoxia, oxygenase activity increased while deaminase activity decreased. The NADPH-dependent electron-transport system necessary for the microsomal oxygenase activity was largely inactivated after the 10th day of hypoxia, while oxygenase activity was not affected. The particulate porphobilinogen oxygenase could be solubilized from the bone marrow microsomes with 1% deoxycholate or 0.5 M KCl. In addition, the oxygenase was also released by freezing and thawing the microsomes isolated from bone marrow of rats which had been submitted to an erythropoietic stress (hypoxia or phenylhydrazine). The enzyme solubilized with deoxycholate or KCl showed a high molecular weight form and a low molecular weight form (Mr 25 000). The former could be transformed into the latter either by treatment with 2 M KCl or by succinylation. When the oxygenase was solubilized by freezing and thawing a third molecular weight form (Mr 50 000) also appeared. The solubilized enzyme could be succinylated without loss of its catalytic activity, while the membrane-bound enzyme could not be succinylated.

Induction of porphobilinogen oxygenase and porphobilinogen deaminase in rat blood under conditions of erythropoietic stress.

Porphobilinogen is the substrate of two enzymes: porphobilinogen deaminase and porphobilinogen-oxygenase. The first one transforms it into the metabolic precursors of heme and the second diverts it from this metabolic pathway by oxidizing porphobilinogen to 5-oxopyrrolinones. Rat blood is devoid of porphobilinogen-oxygenase under normal conditions while it carries porphobilinogen-deaminase activity. When the rats were submitted to hypoxia (pO2 = 0.42 atm) for 18 days, the activity of porphobilinogen-oxygenase appeared at the tenth day of hypoxia and reached the maximum at the 14-16th day. It decreased to a half after 2 days (half-life of the enzyme) and disappeared after 4 days of return to normal oxygen pressure. Porphobilinogen-deaminase activity increased after the first day of hypoxia, reached a maximum at the 14-16th day and did not decrease to normal values until the 15th day after return to normal oxygen pressure. The activities of both porphobilinogen-oxygenase and porphobilinogen-deaminase were induced by administration of erythropoietin. When rats were made anaemic with phenylhydrazine, porphobilinogen-oxygenase activity also appeared in the blood cells. Although the reticulocyte concentration was higher when compared to that obtained under hypoxia, the activities of the oxygenase obtained under both conditions were comparable. Porphobilinogen-deaminase activity was always closely related to the reticulocyte content. The appearance of porphobilinogenase-oxygenase under the described erythropoietic conditions was due to a de novo induction of the enzyme, as shown by its inhibition with actinomycin D and cycloheximide. Porphobilinogen-oxygenase as well as porphobilinogen-deaminase were present in the rat bone marrow under normal conditions. Their activities increased in phenylhydrazine treated rats. The properties and kinetics of porphobilinogen-oxygenase from the rat blood and bone marrow were determined and found it differ in several aspects.

Are beta-adrenoreceptors involved in the intestinal absorption and tissue distribution of iron in the rat?

59Fe was incorporated in vivo into intestinal sacs prepared in iron deficient rats and 59Fe counts were detected in blood, spleen, liver, femur and intestine. The i.v. injection of isoproterenol (i.v. 2 micrograms/rat) or N',O'-dibutyryladenosine 3',5'-cyclic monophosphate (10 microM/100 g b.w.) enhanced significantly the iron counts in blood, spleen, liver and femur but not in intestine. (-)-Propranolol (2 mg X kg-1 b.w. i.v.) antagonized the stimulatory effect of isoproterenol. It is suggested that isoproterenol increases iron absorption via the stimulation of beta-adrenoreceptors of the intestinal mucosa.

Intestinal iron absorption during turpentine sterile inflammation in the rat. Influences of isoproterenol and propranolol.

59Fe was incorporated in vivo into intestinal sacs prepared in control rats as well as in animals with turpentine sterile abscesses and 59Fe counts were detected in the intestinal wall, in blood, in liver, in spleen and in femur of animals, at different time intervals (20, 40 and 120 min) following i.v. injection of isoproterenol (10 micrograms/kg, body weight) or of (-)-propranolol (2 mg/kg, body weight). In rats without inflammation isoproterenol alone enhanced significantly iron counts in blood, spleen, liver and femur, but not in intestinal cells, whereas propranolol evoked opposite actions, the influence being more marked after 40 min, reaching maximal values at 120 min. In animals with sterile turpentine abscesses explored 40 min after injections, iron intestinal counts in turpentine, in turpentine plus isoproterenol and in turpentine plus propranolol groups were significantly lower than in normal saline injected controls (p less than 0.001) and the opposite was the case at 120 min. In blood, iron counts at 20 and 40 min were higher in saline controls (p less than 0.05) than in the turpentine group, whereas at 120 min, values in the turpentine plus isoproterenol group were higher than (p less than 0.01) in saline injected controls or in the group with turpentine alone. Also, at all time intervals explored, iron counts in blood in the propranolol plus turpentine group were smaller (p less than 0.01) than in saline injected controls or in the group with turpentine alone.(ABSTRACT TRUNCATED AT 250 WORDS)

The inhibitory action of aluminum on mouse bone marrow cell growth: evidence for an erythropoietin- and transferrin-mediated mechanism.

Aluminum (Al) has been associated with anemia in chronic renal failure patients under hemodialysis as well as in Al-overloaded animals. In an attempt to elucidate further the mechanism of Al toxicity we have investigated the effect of this ion on erythropoiesis in vitro. Mouse bone marrow cells were stimulated in vitro with erythropoietin (Epo) in the presence of Al3+ ion and erythroid colony-forming units were then determined. Results of this study indicate that Al compounds (chloride and citrate) at concentrations as low as 0.37 mumol Al/l inhibit erythropoiesis in vitro through a mechanism dependent upon the availability of transferrin to bind to aluminum. This process cannot be reversed by increasing Epo doses. This inhibition only occurs in the presence of Epo at early stages during the interaction of the hormone with its target cell.

Depressed erythroid progenitor cell activity in aluminum-overloaded mice.

The aim of the present study was to evaluate the effect of aluminum (Al) on in vivo erythropoiesis in mice that had received short- and long-term Al overloading. At the end of each treatment period, clonal assays of late erythroid progenitor cells (CFU-E) stimulated in vitro with erythropoietin were carried out, hematological parameters determined, and histological aluminon staining performed on bone and liver. After 2 weeks of oral ingestion of either Al chloride or Al citrate (10 mumol per day), poor CFU-E growth was obtained but no differences in hematocrit (Ht) and hemoglobin (Hb) values were found when comparing the treated and control groups. CFU-E development, determined after loading mice with Al citrate during 22 weeks (10 mumol/day), proved to be heavily depressed, and significant reductions in Ht, Hb concentration and erythrocyte osmotic fragility were also observed. No Al accumulation could be demonstrated in tissue, using histological aluminon staining. The results suggest that, even in the absence of signs of anemia, ingested Al may depress hematopoiesis by affecting red blood cell production and cell destruction.

Action of blood serum from rats with turpentine sterile inflammation on the development of CFU erythrocyte colonies. Possible role of erythropoietin.

Bone marrow CFUe mice cultures were prepared in Petri dishes and the number of full developed erythrocyte colonies were counted under various experimental conditions. In certain experiments, erythropoietin (EP = 0.4 U x ml-1 in the suspending medium) or sera from normal rats (100 microliters) or from animals with chronic turpentine sterile abscesses (60 or 100 microliters), were delivered to the CFUe colonies. The number of colonies in the group without EP was almost 5 times smaller than in the group with EP. Inasmuch as some few colonies are still able to develop, even in absence of added EP, the suggestion is advanced holding that the phenomenon may represent the effect of EP already bound to the group of cells initiating differentiation. Comparisons among all experimental groups indicate that the delivery of sera from turpentine rats to cultures containing added EP, reduced significantly the number of CFUe colonies seen in controls with EP but without inflammatory serum. It is suggested that the present findings could be explained assuming an inhibition of the influence of exogenous EP, subserved by the inflammatory serum, or alternatively that this kind of rat serum induces a partial blockade of EP at receptor sites whose activation is a mandatory step for the adequate and full development of cultured erythrocytes.

Erythropoietin modified the cardiac action of ouabain in chronically anaemic-uraemic rats.

The results of the studies reported here demonstrate the cardiac non-haematopoietic effect of erythropoietin, providing a new physiological function of the hormone. We demonstrate that myocardium from rat with chronic renal failure (CRF) showed an abnormal response to ouabain associated with an inhibition of cardiac Na+/K+/ATPase activity and with a decrease in the high affinity 3H-ouabain binding sites. The extent to which both actions were improved with the recombinant human erythropoietin (rHuEpo) treatment suggests that the lack of the hormone is responsible for this phenomenon. The fact is that neither contractile nor enzymatic action of rHuEpo was accompanied with the improvement of the functional renal and haematologic parameters, indicating a primary effect on myocardial contractile function of rHuEpo, independent of the anaemic and uraemic state of the animal. The reason why erythropoietin is able to modulate directly the cardiac Na+/K+ pump makes it possible to conclude that the lack of erythropoietin in CRF may be at least in part responsible for the inhibition of cardiac enzymes, altering the contractile behaviour of the heart.

The inhibitory action of chlorogenic acid on the intestinal iron absorption in rats.

The polyphenols are part of the composition of many foods, it is known the inhibitory effect of tea and coffee through the tannins on iron intestinal absorption; the "yerba mate" (Ilex Paraguarensis) is a beverage widely used in South America, that has a high content of a polyphenol named chlorogenic acid. The present work shows the effect of this substance in nonhem iron absorption. An intestinal loop, was made in rats, to form a closed cavity in a small section of intestine tieing it from the pilorous to a distance of six cm. In this closed cavity a solution of 59Fe was injected with different doses of chlorogenic acid; it was living 20, 40 and 120 minutes into the loop, and after this different times, the blood, spleen, liver, femur and intestine were removed to measure the 59Fe uptake to be compared with the control group. The results gave an intense inhibitory effect on the intestinal iron absorption with doses of 0.58 and 1.7 mM per rat of chlorogenic acid at the different times studied.

Effect of acetylsalicylic acid on iron absorption in the rat.

The in vivo administration of 59Fe to the rat accompanied by acetylsalicylic acid (ASA) enhanced significantly counts in blood, spleen, liver and femur without affecting those of the intestine. The results suggest that ASA augments iron absorption either via an inhibitory action on the synthesis of prostaglandins or by a purely chemical mechanism.

Direct evidence favouring the notion that erythropoietin alters iron transport across the isolated intestinal tract of the rat.

A simple and reproducible method, using the isolated not everted intestine of the rat, for the study of iron transport is presented. Erythropoietin (ESF) enhanced significantly the passage of 59Fe across the intestine augmenting its movement at mucosal and serosal layers of the intestinal well.

Myocardial mitogenic effect of erythropoietin through the activation of Na(+)-K(+)-ATPase activity.

Erythropoietin is considered unique among the hematopoietic growth factor with a specific action on the differentiation and proliferation of erythroid progenitor cells. We have observed a dose-dependent modulatory action of human recombinant erythropoietin (rHuEpo) stimulated the rate of cell growth but at higher ones (3-10 U/ml) inhibited it. The mitogenic action of the hormone is correlated with cardiac membrane Na(+)-K(+)-ATPase activity since concentrations of rHuEpo that increased cell growth stimulated paranitrophenilphosphatase (pNPPase) activity, while those concentrations that inhibit the enzyme markedly bloqued its mitogenic action. Moreover, ouabain (10(-5) M), concentration that inhibits Na(+)-K(+)-ATPase activity, blunted the stimulatory action of rHuEpo on cell proliferation. We also demonstrated that rHuEpo while activated the cardiac membrane Na(+)-K(+)-ATPase was able to alter the contractile action of ouabain on isolated neonatal rat atria. Indeed rHuEpo (1 U/ml) enhanced the non toxic action of the cardiac glycoside attenuating and delaying the onset of the toxic effect of the drug. These results show that rHuEpo has a non hematopoietic cardiac effect, associated with the cardiac Na(+)-K(+)-ATPase activity, that regulates the myocytes growth and the biological action of cardiac glycosides on isolated rat myocardium.

Effect of 15(R)-15-methyl-prostaglandin E2 on iron absorption in the rat.

The administration of 15(R)-15-methyl prostaglandin E2 (15(R)-15-M-PGE2) in vivo significantly diminished the uptake of (59)Fe into blood, spleen, liver, femur and dried intestine of rats, whereas acetylsalicylic acid (ASA) increased the counts significantly. This effect of ASA was counteracted by 15(R)-15-M-PGE2. It is suggested that prostaglandins (PGs)might play an important role in inhibiting iron absorption at the intestinal level.