Studies of the Influence of Cyclic Nucleotides on in Vitro Haemoglobin Synthesis

Abstract

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.

Modulation of in vitro erythropoiesis. The influence of beta-adrenergic agonists on erythroid colony formation.

“…First, while cAMP enhanced colony growth, only ESF was capable of initiating erythroid colony formation (3). Second, the adenyl cyclase stimulator, cholera enterotoxin, continued to augment colony formation (3), and cAMP continued to induce hemoglobin synthesis in cell cultures (2) …”

“…Since adenyl cyclase-linked mechanisms regulate differentiation, growth, and function in a number of tissues, such mechanisms may also influence erythropoiesis. Evidence that selected cyclic nucleotides may stimulate mammalian erythropoiesis has been provided by in vitro studies demonstrating that cyclic AMP (cAMP) increases canine hemoglobin synthesis in marrow suspension culture (2) and enhances ESFdependent erythroid colony formation in semisolid media (3). Since the presence of ESF is essential for colony formation, and since the cells most responsive to cAMP or ESF are physically and functionally dissimilar, it has been proposed that mechanisms linked to cyclic nucleotides may modulate in vitro ESF action (4).…”

Extracellular cAMP inhibits proximal reabsorption: are plasma membrane cAMP receptors involved?

“…Finally, extracellular cAMP is suspected to play a role in the regulation of erythropoiesis. cAMP and several analogs have been shown to stimulate erythropoiesis in plethoric mice (132,155) and hemoglobin synthesis in bone marrow cells from humans, dogs, sheep, and rabbits, but not in cells isolated from rodents (32). However, a rise in hemoglobin, hematocrit, and red cell mass was also observed in mice after chronic dibutyrylcAMP treatment (144).…”

Stimulation of Newborn-Like Hemoglobin Synthesis in Adult Rats by Recombinant Human Erythropoietin and Suppression by Aspirin

“…Our previous work demonstrated that indomethacin, like aspirin, also totally blocked the bleedinginduced reversion o f hemoglobin proportions toward newborn values in adult rats. This finding prompted us to rule out the possibility that aspirin could acetylate many proteins and thereby could exert pharmacologic actions unrelated to its effect on PG biosynthesis [36], However, on the basis o f the present study it is not possible to offer a specific mechanism o f action for the need o f ongoing PG synthesis in order to achieve the reversion o f Hb propor tions in adult rats, other than the already known general informations that PGs exert their effect locally and many o f the effects o f these compounds are mediated by 3',5'-cyclic AMP (cAMP) [38], Earlier studies have indicated that erythropoietin action is mediated by cAMP [39,40], a concept further strengthened by the observation that the bone marrow cAMP level is significantly elevated in phenylhydrazine-induced anemic rats [41]. It remains to be seen whether concurrent PG synthesis is required to regu late the synthesis o f cAMP and whether such a situation is necessary for the reverse switching process.…”