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 μM 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 β-agonist l-isoprenaline. The in vitro effect of erythropoietin is abolished by the β-thio analogue of GDP, GDP[βS], 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 nucleotide-binding protein with high affinity for GTP (EC50 < 10 nM), whereas haemin appears to activate adenylate cyclase indirectly, as a consequence of progressive perturbations of the plasma membrane.
effective concentration for half-maximum activity
Adenylate cyclase, ATP pyrophosphate-lyase (cyclizing) (EC 18.104.22.168)