Classification of beta-adrenergic subtypes in immature rabbit bone marrow erythroblasts.

The beta-adrenergic receptors of immature rabbit bone marrow erythroid cells (proerythroblasts and basophilic erythroblasts) were identified. [125I]iodocyanopindolol bound to membrane preparations derived from these erythroblasts in a rapid, reversible and saturable manner. Scatchard analysis of binding data revealed a single class of binding sites (Hill coefficient of 0.954) with an apparent equilibrium dissociation constant (Kd) of 8 pM, and a density of binding sites (Bmax) of 1.53 pM/10(6) cells, corresponding to 920 receptors per cell. The binding of [125I]iodocyanopindolol was inhibited stereospecifically by concentrations of (-)-propranolol 2 orders of magnitude lower than by the (+)-isomer. Only L-isoprenaline and L-adrenaline activated the adenylate cyclase of immature rabbit erythroblasts, while L-noradrenaline, a beta 1-adrenergic agonist, was inactive. The order of potency of different agonists for displacement of bound [125I]iodocyanopindolol was: isoprenaline greater than adrenaline greater than noradrenaline with respective EC50 (concentration required for half maximal inhibition of binding) of 7.9 X 10(-7) M, 1.5 X 10(-5) M and 7.9 X 10(-5) M. This agonist potency series did not change with differentiation of rabbit bone marrow erythroblasts. The inhibition of specific [125I]iodocyanopindolol binding to immature cells by beta 1- and beta 2-selective drugs (noradrenaline, practolol, procaterol and butoxamine) resulted in linear Hofstee plots. The inhibition curves obtained with procaterol and butoxamine, with apparent Kd values of 3.1 X 10(-9) M and 4.9 X 10(-9) M, further evidence that the high-affinity binding sites correspond to a homogeneous beta 2-receptor subtype.

Independent activation of adenylate cyclase by erythropoietin and isoprenaline.

The possibility that catecholamines modulate the erythropoietin-induced increase in production of cyclic AMP was investigated by examining the effect of erythropoietin and/or L-isoprenaline on the activity of the plasma membrane adenylate cyclase of anaemic rabbit bone marrow erythroblasts. Membranes isolated from cells cultured in the presence of both hormones exhibited both the transient stimulation of basal activity characteristic of erythropoietin action and the loss of the in vitro response to L-isoprenaline, concomitant with the loss of beta-adrenergic receptors, characteristic of L-isoprenaline stimulation. The presence of erythropoietin during cell culture with L-isoprenaline had no effect on the desensitization or number of beta-adrenergic receptors. The stimulation of adenylate cyclase by erythropoietin was observed also in the presence of the beta-antagonist propranolol, when both were added either to whole cells or to isolated membranes. We conclude that these two hormones activate adenylate cyclase independently of each other, via different receptors, with little evidence of cross-modulation.

Regulation of erythroid cell differentiation by haemin.

The differentiation of immature erythroblasts, isolated from anaemic rabbit bone marrow by density centrifugation to bovine serum albumin gradients, is accelerated by the addition of 10(-5)-10(-4) M haemin to the culture medium. Both the proportion of benzidine-positive cells and the synthesis of haemoglobin relative to the total protein were increased, whereas cell growth and DNA synthesis were decreased. Some of these changes were detected within 4 h and were maximal after 18-40 h. It is suggested that haem may have a physiological role in regulating in vivo erythropoiesis during haemolysis by accelerating terminal erythroid cell differentiation.

Subcellular localization of a lesion in protein synthesis in rabbit reticulocytes incubated at elevated temperatures.

When rabbit reticulocytes are incubated at 43-45 degrees C their rate of protein synthesis rapidly decreases, compared to a contol 37 degrees C incubation. Lysates prepared from cells incubated at this supra-optimal temperature have an equally decreased capacity for endogenous, but not poly(uridylic acid)-directed, protein synthesis. Subcellular fractionation traced the lesion to the crude ribosomal pellet, 0.5 M KCl ribosomal wash and postribosomal supernatant of the temperature-shocked cells. Preparation of purified ribosomal subparticles showed, however, that they were as active as the control in protein synthesis. In this paper we present evidence that the decreased activity of the heated lysate, 0.5 mM KCl wash and postribosomal supernatant is due to an inhibitor and can be overcome by the addition of 0.5 M KCl or supernatant from control cells. The results are discussed in terms of the inactivation of a component, essential for initiation of endogenous protein synthesis, which is probably partitioned between ribosomes and supernatant. We also suggest that the decreased protein synthetic activity of the heated cells may be related to their decreased synthesis of haem.

Protein phosphorylation in erythroid cell development.

Changes in the phosphorylation of proteins during erythroid cell development have been investigated by assaying the activity of three protein kinases in circulating reticulocytes, and dividing and non-dividing erythroblasts obtained from the bone marrow of anaemic rabbits. Kinase activities decreased during erythroid cell development, but protein phosphorylation was generally limited by substrate availability rather than enzyme activity. Using permeabilized cells some changes in the patterns of proteins phosphorylated by [gamma-32P]ATP were observed during erythroid cell development.

Regulation of protein synthesis in reticulocyte lysates. Characterization of the inhibitor generated in the postribosomal supernatant by heating at 44 degrees C.

Heating of a rabbit reticulocyte lysate at 44 degrees C, in the presence of optimal concentrations of haemin, results in an inhibition of protein synthesis and in the appearance of an inhibitory activity in the postribosomal supernatant. These effects of supraoptimal heating are similar to those observed in lysates and supernatants incubated at physiological temperatures in the absence of haemin. In this paper we examined whether the haem-regulated inhibitor produced by treating postribosomal supernatant with N-ethylmaleimide and the inhibitor generated by heating at 44 degrees C in the presence of haemin are the same molecular entity. Both inhibitors behaved similarly through a partial purification consisting of 50% (NH4)2SO4 precipitation, Sephadex G-150 gel filtration and DEAE-cellulose chromatography, and had the same pattern of polypeptides after polyacrylamide gel electrophoresis. However, when incubated with an antiserum to the haem-regulated inhibitor the activity of the 44 degrees C heated supernatant was not neutralized, whereas that of its more purified fractions was. This apparent contradiction was shown to be due to an interference of the immune serum assay by the levels of proinhibitor and haemoglobin present in the crude supernatant. Further experiments, with extensively diluted 44 degrees C supernatants or with isolated proinhibitor subsequently heated, are consistent with the conclusion that both heating at supraoptimal temperatures and incubating in the absence of haem finally activate the same inhibitor.

The role of cAMP and calcium in the stimulation of proliferation of immature erythroblasts by erythropoietin.

The hypothesis that cAMP or calcium are the second messengers of erythropoietin (Epo) was tested on fractionated, Epo-responsive immature erythroblasts from anemic rabbit bone marrow by examining whether the proliferative effects of the hormone could be mimicked by agents that increase the intracellular concentration of cAMP or Ca2+. None of the compounds tested (including 10(-6)-10(-4) M db-cAMP, forskolin, isoprenaline or 10(-7)-10(-6) M of the calcium ionophore A23187) alone or in combination could either initiate or potentiate the mitogenic action of the hormone. Furthermore, addition of 0.2 U/ml erythropoietin produced no permanent or transient increase in the uptake of 45Ca2+ by erythroblasts at 37 degrees C. However, cells cultured with imidazole or cordycepin (which reduce the level of intracellular cAMP), or with the calcium chelator EGTA, or the drugs verapamil or TMB-8 (which interfere with the utilization of extracellular or intracellular calcium) showed a decreased stimulation of DNA synthesis by Epo. Finally, the tumour promoter phorbol ester TPA could partially mimic the action of Epo when added to cultures containing more immature progenitor cells. We conclude then that an artificial increase in the cytoplasmic concentration of either cAMP or Ca2+ is not sufficient to elicit the proliferation of Epo-responsive cells.

Stimulation of the adenylate cyclase activity of rabbit bone marrow immature erythroblasts by erythropoietin and haemin.

The effect of two agents of erythroid cell differentiation on the adenylate cyclase activity of fractionated rabbit bone marrow erythroblasts has been investigated. Addition of 0.2U/ml erythropoietin to cell cultures causes a transient increase in the activity of plasma membrane adenylate cyclase, which is maximal by 20 min and disappears within 4 h. The magnitude of the response to hormonal stimulation depends on the stage of erythroid cell development and is greater in the more immature cells. Addition of 50 microM haemin to cultures of erythroblasts also causes an increase in the activity of adenylate cyclase, which differs from the effect of erythropoietin in kinetics and specificity of target cells. With immature cells the haemin-induced stimulation starts after the first hour and continues to increase up to 20 h of culture. Erythropoietin but not haemin can stimulate the basal activity of adenylate cyclase in an in vitro assay containing plasma membranes of immature erythroid cells. The degree of activation depends on the concentration of erythropoietin and is maximal with 0.2-0.5 U/ml hormone (5-12 nM). In the presence of guanine nucleotides the activation of adenylate cyclase by erythropoietin is increased further but the effect is not additive. With respect to the basal and the guanine-nucleotide-stimulated activities of adenylate cyclase erythropoietin acts differently from the beta-agonist l-isoprenaline. The in vitro effect of erythropoietin is abolished by the beta-thio analogue of GDP, GDP[beta S], and extensive washing of membranes makes hormone action GTP-dependent. The stimulation of adenylate cyclase by the addition of erythropoietin to the reaction mixture is inversely related to the extent of previous hormonal stimulation of the cells from which the membranes were prepared. This loss of hormonal responsiveness is due to desensitization or receptor down-regulation and persists for up to 20 h. We conclude that in immature erythroblasts erythropoietin acts via a receptor and a guanine nucleotide-binding protein with high affinity for GTP (EC50 less than 10 nM), whereas haemin appears to activate adenylate cyclase indirectly, as a consequence of progressive perturbations of the plasma membrane.

The effect of erythropoietin on the adenylate cyclase activity of rabbit bone marrow erythroblasts.

The involvement of adenylate cyclase in the response elicited by erythropoietin was investigated in fractionated erythroblasts obtained from anaemic rabbit bone marrow. Addition of 0.2 U/ml erythropoietin to cell cultures caused a transient increase in the activity of plasma membrane adenylate cyclase, which was observed within 5 minutes, was maximal by 20 minutes and disappeared within 4 hours. The magnitude of the response to hormonal stimulation depended on the stage of erythroid cell development and was greater in the more immature cells. Erythropoietin could also stimulate the basal activity of adenylate cyclase in an in vitro assay containing plasma membranes of immature, but not mature, erythroid cells. The degree of activation was hormone-concentration dependent, was maximal at 0.2-0.5 U/ml erythropoietin (5-12 nM) and was observed in the absence of exogenous guanine nucleotides. The in vitro effect of erythropoietin, however, was abolished by GDP (S) and extensive washing of the membranes made hormone action GTP-dependent. The ability of the hormone to stimulate adenylate cyclase activity in vitro was inversely related to the extent of hormonal stimulation in vivo. This desensitization was observed within 20 minutes and persisted for many hours. It is suggested that erythropoietin activates the adenylate cyclase of immature erythroblasts via a receptor and a guanine nucleotide-binding protein with high affinity for GTP.

The effect of haemin on RNA synthesis and stability in differentiating rabbit erythroblasts.

Haemin accelerates the maturation of erythroid cells but whether this is the result of increased globin gene transcription or processing and stabilization of globin mRNA is not clear. The effect of haemin on the synthesis and stability of non-globin messengers is also unknown. We examined the changes that occur in RNA metabolism when anaemic rabbit bone marrow erythroblasts, fractionated into immature and mature fractions, are cultured with 20 microM or 50 microM haemin for brief periods (5-8 h). With both cell types haemin increases the incorporation of [3H]uridine into newly synthesized RNA, particularly into the poly(A)-rich fraction which can increase threefold. Haemin also increases the synthesis of globin mRNA (up to 500% absolutely and 50% relative to the synthesis of total RNA) in the immature, but not in the mature, cells. These results suggest that haemin increases the transcription of both globin and non-globin mRNAs and that the relative increase of each depends on the stage of erythroid cell development. When the [3H]RNA from prelabelled cells was chased in the presence of haemin (with or without actinomycin D) the proportion of 3H remaining in globin mRNA increased in the immature, but not in the mature, cells. These changes in the relative concentration of globin mRNA were also shown by the translation of extracted bone marrow RNAs in a nuclease-treated reticulocyte lysate. We conclude that a secondary effect of haemin is on RNA stability and that it enhances the accumulation of globin mRNA by both molecular and cellular mechanisms.