STIMULATION BY ENDOTHELIN-1 OF MITOGEN-ACTIVATED PROTEIN-KINASES AND DNA-SYNTHESIS IN BOVINE TRACHEAL SMOOTH-MUSCLE CELLS

1 In cultures of bovine tracheal smooth muscle cells, platelet-derived growth factor-BE (PDGF), bradykinin (BK) and endothelin-1 (ET-1) stim ulated the tyrosine phosphorylation and activation of both pp42 and pp 44 kDa forms of mitogen-activated protein (MAP) kinase. 2 Both ET-1 an d PDGF stimulated a sustained activation of MAP kinase whilst the resp onse to BK was transient. 3 Activation of MAP kinase occurred in a con centration-dependent manner (EC(50) values: ET-1, 2.3 +/- 1.3 nM; BK, 8.7 +/- 4.1 nM, PDGF, 9.7 +/- 3.2 ng ml(-1)). 4 Pretreatment with the protein kinase C (PKC) inhibitor Re-318220, significantly reduced ET-1 activation of MAP kinase at 2 and 5 min but enhanced MAP kinase activ ation at 60 min. 5 Following chronic phorbol ester pretreatment, BK-st imulated activation of MAP kinase was abolished whilst the responses t o PDGF and ET-1 were only partly reduced (80 and 45% inhibition respec tively). 6 Pretreatment with pertussis toxin reduced ET-1 stimulated a ctivation of MAP kinase particularly at later times (60 min), but left the responses to both PDGF and BK unaffected. 7 ET-1 also stimulated a 3 fold increase in [H-3]-thymidine incorporation which was abolished by pertussis toxin pretreatment. In contrast, PDGF stimulated a 131 f old increase in [3H]-thymidine incorporation which was not affected by pertussis toxin. 8 These results suggest that a pertussis toxin-sensi tive activation of MAP kinase may play an important role in ET-1-stimu lated DNA synthesis but that activation of MAP kinase alone is not suf ficient to induce the magnitude of DNA synthesis observed in response to PDGF.