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

Abstract

The effect of two agents of erythroid cell differentiation on the adenylate cyclase activity of fractionated rabbit bone marrow erythroblasts has been investigated.1. Addition of 0.2 U/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.2. Addition of 50 pM 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.3. 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 B-agonist I-isoprenaline. The in vitro effect of erythropoietin is abolished by the B-thio analogue of GDP, GDP [PS], and extensive washing of membranes makes hormone action GTP-dependent.4. 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.5. We conclude that in immature erythroblasts erythropoietin acts via a receptor and a guanine nucleotidebinding protein with high affinity for GTP (EC5,, < 10 nM), whereas haemin appears to activate adenylate cyclase indirectly, as a consequence of progressive perturbations of the plasma membrane.The glycoprotein hormone erythropoietin regulates the proliferation and differentiation of erythroid cells [l, 21.Haemin is one of a number of other compounds which also have a regulatory effect on the maturation of erythroleukaemic [3] or normal [4-61 erythroblasts. The mode of action of either erythropoietin or haemin is not clear. Many glycoprotein hormones elicit a biological response by activation of the adenylate cyclase system [7] and in eukaryotes cAMP can act as a mitogenic signal and can induce the transcription of specific genes [8]. There is no general agreement in the literature, however, concerning the involvement of cyclic nucleotides in the response elicited by erythropoietin or in erythropoiesis in general. Addition of erythropoietin was reported to cause a biphasic increase in cAMP levels in rab...

Erythropoietin and testicular steroidogenesis: the role of second messengers

“…that have been shown to influence Leydig cells steroidogenesis. Different molecular events follow the binding of EPO to its cell surface receptors (15)(16)(17)(18)(19)38). Early studies of the mechanism of action of erythropoietin addressed the possibility that cyclic nucleotides might serve as second messengers (18,19).…”

“…In different cellular systems, several biochemical processes have been shown to participate in erythro¬ poietin signal transduction, including protein phos¬ phorylation (15)(16)38), changes in intracellular calcium concentration (17) and activation of second messengers as well as cyclic nucleotides, inositol 1,4,5-trisphosphate and diacyglycerol (18,19). In light of these observations, the present study was carried out to evaluate the intracellular events involved in recombi¬ nant human erythropoietin (rHuEPO)-stimulated steroidogenesis in adult rat Leydig cells.…”

Coupling of Heterotrimeric Gi Proteins to the Erythropoietin Receptor

“…Some studies have suggested that Epo may increase adenylate cyclase activity and cAMP levels (40,68), whereas others did not report any modification of cAMP (69). However, to our knowledge, no studies have reported a decrease in cAMP levels in response to Epo stimulation.…”

Induction of tyrosine phosphorylation by the erythropoietin receptor correlates with mitogenesis.

“…The signaltransducing mechanisms in these responses are largely unknown, in part because of the difficulty of obtaining pure populations of Epo-dependent cells. Results of several studies using heterogenous populations of hematopoietic cells have suggested a role for cyclic AMP or changes in calcium levels (1,27,28,36). However more recent studies, using highly enriched or pure populations of Epo-responsive cells, have failed to support a role for these mediators in proliferation (24).…”