In intact reticulocytes, but not in fragmented membranes, the loss of adenylate cyclase activity during cell maturation followed a biphasic time course. A rapid phase (t1/2 approximately 2 h) during which the initial activity was reduced by 40-50% was followed by a slow phase with t1/2 close to 3 days. The fast decay seemed to occur on the adenylate cyclase level since (-)isoprenaline- or forskolin-stimulated activities behaved similarly and bacterial toxin-monitored Gs and Gi proteins remained stable. The mechanism of the initial decrease in hormonal responsiveness was further analysed in hybrid cells prepared by fusing reticulocytes with Friend erythroleukemia (MEL) cells. The hybrids contained reticulocyte-derived beta-adrenoceptors and MEL cell-derived adenylate cyclase and G proteins. Fusion of reticulocytes to native MEL cells caused adenylate cyclase activity to drop by 30% at 2 h and 45% at 18 h after fusion. By contrast, hybrids prepared after dimethylsulfoxide-induced differentiation of MEL cells showed stable or increasing rates of receptor-coupled cAMP formation between 2 and 18 h after fusion, concomitant with the enhanced activity of the Gs protein in these cells. A cyclase-stimulating factor present in the cytosol of MEL cells and of reticulocytes appeared not to be involved in short-term regulation of hormonal responsiveness. We conclude that the strength of beta-adrenergic responses in erythroid progenitor cells is primarily regulated by modulating G protein-mediated receptor cyclase coupling while reticulocytes, during early maturation, seem to rely on direct inactivation of adenylate cyclase, probably via a cytosolic proteolytic pathway.

The loss of beta-adrenergic responsiveness during reticulocyte maturation was studied under tissue culture conditions in a defined cell population from rats. Initial beta-receptor density and receptor-mediated cAMP formation in culture medium (RPMI 1640) exceeded the values obtained in isotonic KC1-buffer by 56 and 120% respectively. During cell cultivation receptor density and hormonal responsiveness decreased rapidly by 40-50% of their initial values within the first 20 h. In the following 3 days the rate of loss varied between 10 and 15%/d. The same time course was observed for the maturation-dependent decrease in forskolin-stimulated cAMP formation. ATP depletion of cultivated cells caused a complete and irreversible loss of cAMP response within 90 min. Our results indicate that cell metabolism regulates the strength of the hormonal response. A defect in adenylate cyclase or in cyclase N-protein interaction seems to be rate-limiting for the functional inactivation of the beta-adrenergic system during reticulocyte maturation.

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