CA2-CELLS - EFFECT OF DIMETHYL-SULFOXIDE AND ROLE OF G-PROTEINS IN THROMBIN AND THROMBOXANE A(2)-ACTIVATED PATHWAYS( SIGNALING IN K562 HUMAN ERYTHROLEUKEMIA)

The human leukaemic cell line K562 is a pluripotent stem cell with the potential to mature along a megakaryocytic or erythroid line. In thes e cells, thrombin and U46619 (9,11-dideoxy-9 alpha,11 alpha-methanoepo xy prostaglandin F-2 alpha), a thromboxane A(2) analogue, increased in tracellular Ca2+ in a rapid and concentration-dependent manner. The pe ak transient observed with both thrombin and U46619 was preserved upon stimulation in the absence of extracellular calcium and blunted with phorbol myristate acetate, Suggestive of activation of phospholipase C . Short-term treatment with leupeptin abolished the calcium response t o thrombin, but did not alter that to U46619. Both pertussis toxin (PT ) and DMSO pretreatment inhibited thrombin- but not U46619-stimulated intracellular calcium elevation, indicating that these agonists signal through different G-proteins. Western blot analysis of crude membrane s from K562 cells revealed the presence of G(i2)alpha and G(i3)alpha; the other known PT-substrates, G(i1)alpha. and G(o) alpha, were not de tected. Consistent with this observation, ADP-ribosylation experiments revealed the presence of two PT substrates which co-migrated with hum an erythrocyte G(i2)alpha and G(i3)alpha. An antibody raised against G (q/11)alpha, a subfamily of G-protein alpha subunits unmodified by PT, specifically recognized 42 kDa protein(s) in K562 cells. PCR amplific ation of reverse-transcribed K562 RNA followed by DNA sequencing showe d that these cells express messages for both G(q) alpha and G(11)alpha . Treatment of K562 cells with DMSO reduced the levels of thrombin rec eptor mRNA, without simultaneous changes in the expression of G(i2)alp ha and G(i3)alpha. We have thus identified Ca2+-mobilizing agonists an d related G-proteins in K562 cells, together with changes induced by D MSO in this signalling pathway.