Cyclic AMP Maintenance of Rabbit Reticulocyte Haem and Protein Synthesis in the Presence of Ethanol and Benzene

Abstract

SUMMARY.Haemin is necessary for maximal protein synthesis in intact reticulocytes and their cell‐free lysate preparations. Under conditions of haemin deficiency a translational protein repressor of initiation (HCRA) forms from a non‐inhibitory form of the same protein (HCR1) and protein synthesis ceases. It has recently been suggested that this repressor is a protein kinase and that high concentrations of 3′,5’cyclic AMP could substitute for haemin in maintaining reticulocyte lysate cell‐free protein synthesis. The present study was undertaken, therefore, to investigate if cAMP had a similar function in intact rabbit reticulocytes. The cells were incubated with eidier epinephrine (10‐6 M), isoproterenol (10‐4 M), theophylline (10‐4 M) or dibutyryl cAMP (10‐4 M). All these compounds elevated the intracellular concentration of cAMP within the first 2 min of incubation, as measured by a specific competitive binding assay. This indicates that rabbit reticulocytes contain both adenyl cyclase and phosphodiesterase systems. The cells were incubated with an inhibitor of haem synthesis (either 0.1 M ethanol or 0.1 M benzene) to form HCRA prematurely and inhibit protein synthesis. When the cells were incubated simultaneously with any one of these inhibitors and either epinephrine, isoproterenol, theophylline or dibutyryl cAMP, there was no inhibition of haem or protein synthesis and no demonstrable HCRA. Furthermore, dibutyryl cAMP reversed the inhibition of both haem and protein synthesis induced by ethanol or benzene. Dibutyryl cAMP relieved the inhibition of haem synthesis prior to relieving the inhibition of protein synthesis, which indicates that sufficient haemin must be present before maximal protein synthesis may resume. In contrast, elevation of cyclic AMP levels did not protect against iron deficiency inhibition of either haem or protein synthesis. This indicates that in intact cells cAMP does not directly convert HCRA to HCR1On the basis of these findings, we postulate that in intact reticulocytes (in contrast to cell‐free systems) a major role of cAMP is maintenance of maximal haem synthesis, even in the presence of inhibitors. Intracellular haemin concentration appears to be a major control mechanism of protein synthesis via prevention and reversal of HCRA. This model assures a balance between haem and globin synthesis in erythroid precursors.