MATURATION OF MEGAKARYOBLASTIC CELLS IS ACCOMPANIED BY UP-REGULATION OF G(S)ALPHA-L SUBTYPE AND INCREASED CAMP ACCUMULATION

In platelets and megakaryoblasts G(s), the trimeric G-protein that sti mulates adenylyl cyclase, is present in a short. 45 kDa, and a long, 5 2 kDa isoform termed G(s) alpha-S and G(s) alpha-L, respectively. To a ssess the relative contribution of these isoforms in the cellular synt hesis of cAMP, the ratio G(s) alpha-S/G(s) alpha-L was enhanced in the megakaryoblastic cell line DAMI by inducing cell maturation with reco mbinant human thrombopoietin (TPO) or the phorbol ester PMA. Flow cyto metric 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 downregula tion of total G(S)-mRNA after treatment of DAMI-cells with TPO and PMA . Western blot analysis showed significant (P <0.05) upregulation of G (s)-L with respective amounts of 27 +/- 4% of total G(s) 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 no t 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 w ith I mu mol/l of the stable prostacyclin analog iloprost. The same st imulation 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 megakaryo blast maturation strongly potentiates the production of cAMP. A simila r shift may occur during normal megakaryocyte maturation and may expla in the extreme sensitivity to prostacyclin of platelets, which contain G(s) alpha-S and G(s) alpha-L in approximately equal amounts.