In platelets and megakaryoblasts Gs, the trimeric G-protein that stimulates adenylyl cyclase, is present in a short, 45 kDa, and a long, 52 kDa isoform termed G(s)alpha-S and G(s)alpha-L, respectively. To assess the relative contribution of these isoforms in the cellular synthesis of cAMP, the ratio G(s)alpha-S/G(s)alpha-L was changed in the megakaryoblastic cell line DAMI by inducing cell maturation with recombinant human thrombopoietin (TPO) or the phorbol ester PMA. Flow cytometric analysis confirmed that this treatment induced a moderate (TPO) and extensive (PMA) increase in nuclear ploidy and expression of the glycoproteins-IIIa and -Ib. Northern blot analysis revealed downregulation of total Gs-mRNA after treatment of DAMI-cells with TPO and PMA. Western blot analysis showed significant (P < 0.05) upregulation of Gs-L with respective amounts of 27 +/- 4% of total Gs in untreated cells, 35 +/- 1% in TPO- and 41 +/- 3% in PMA-treated DAMI cells (n = 3-4). DAMI cells contained 6 +/- 1 pmol cAMP/10(6) cells, which was not changed by treatment with TPO or PMA. In untreated cells this level increased to 70 +/- 9 pmol cAMP/10(6) cells after 10 min stimulation with 1 micromol/l of the stable prostacyclin analog iloprost. The same stimulation with iloprost resulted in 165 +/- 32 pmol cAMP/10(6) in TPO-treated cells and in 588 +/- 100 pmol cAMP/10(6) in cells treated with PMA. Thus, a shift from G(s)alpha-S to G(s)alpha-L during megakaryoblast maturation strongly potentiates the production of cAMP. A similar shift may occur during normal megakaryocyte maturation and may explain the extreme sensitivity to prostacyclin of platelets, which contain G(s)alpha-S and G(s)alpha-L in approximately equal amounts.