Studies of the influence of cyclic nucleotides on in vitro haemoglobin synthesis.

The influence of various cyclic nucleotides on in vitro haemoglobin synthesis has been examined in suspension cultures of mammalian marrow cells. Over a wide range of concentrations, dibutyryl cyclic AMP (db-cAMP) was either ineffective or inhibited haemoglobin synthesis by marrow cells from rat, mouse and guinea-pig. However, 10(-3) M db-cAMP consistently stimulated haemoglobin synthesis in cultures of human, sheep, rabbit and canine cells, with the latter being most responsive. This effect, which approached in magnitude that of erythropoietin (ESF) itself, was specific for cAMP and its mono- and dibutyryl derivatives and was not inhibited by anti-ESF. Adenosine, AMP, ADP, ATP, cGMP, db-cGMP, cCMP, cIMP and sodium butyrate were either inactive or inhibitory at similar concentrations. Enhancement of haemoglobin synthesis was also observed with the phosphodiesterase inhibitor, RO-20-1724. The susceptibility to ionizing radiation of the response to ESF and db-cAMP was marked, indicating that the increased haemoglobin synthesis in this system was proliferation dependent, although the response to db-cAMP was less radiosensitive. Studies with tritiated thymidine showed that about 50% of the cells which were responding to either db-cAMP or ESF were actively engaged in DNA synthesis. However, the physical characteristics of db-cAMP-and ESF-responsive cells were dissimilar as analysed by their velocity sedimentation properties. These studies demonstrate that cAMP has a major stimulatory effect on haemoglobin synthesis with cells from selected mammalian species with activity approaching that of ESF, but the target cells most responsive to these agents appear different. The results suggest that cyclic nucleotide-related mechanisms may modulate in vitro erythropoiesis.