PARALLEL CONTRACTILE SIGNAL-TRANSDUCTION PATHWAYS ACTIVATED BY RECEPTORS FOR THROMBIN AND EPIDERMAL GROWTH FACTOR-UROGASTRONE IN GUINEA-PIGGASTRIC SMOOTH-MUSCLE - BLOCKADE BY INHIBITORS OF MITOGEN-ACTIVATED PROTEIN KINASE-KINASE AND PHOSPHATIDYL-INOSITOL 3'-KINASE

Using a guinea pig gastric longitudinal smooth muscle preparation, we have compared the contractile signaling pathways triggered by the thro mbin receptor-activating peptide, TFLLR-NH2 (TF) and by epidermal grow th factor-urogastrone (EGF). In addition to inhibitors of tyrosine kin ase [tyrphostin 47/AG213, genistein and the src-selective inhibitor CP 118,556/PP1], cyclooxygenase (indomethacin, INDO) and diacylglycerol l ipase (U57, 908), we also used the signal pathway probe inhibitors of mitogen-activated protein-kinase-kinase (MEK:PD98059), phosphatidylino sitol 3'-kinase [Pl3K: Wortmannin (WM) and LY294002], protein kinase C [PKC: GF109203X(GF)], and of the EGF-receptor kinase (PD153035), We f ound that in addition to the inhibition of both TF and EGF-stimulated contractions by the inhibitors of tyrosine kinase, cyclooxygenase and diacylglycerol lipase, the actions of TF and EGF were also attenuated by PD98059, WM/LY294002 and GF. However, PD153035 blocked only EGF-tri ggered contractions. The contractile actions of both TF and EGF were d ependent on extracellular calcium. In contrast, the contractile action of arachidonic acid, via a presumed cyclooxygenase product that media ted the contractions caused by both TF and EGF, was not blocked by any of the signal pathway probe inhibitors. The contractile actions of bo th TF and EGF were accompanied by increases in tissue phosphotyrosyl p roteins and an increase in tissue c-src kinase activity. We conclude t hat protease-activated receptor no. 1-(thrombin receptor) mediated con tractions in the logitudial muscle, like EGF receptor-activated respon ses, require the influx of extracellular calcium and use parallel sign al pathways upstream of the cyclooxygenase step, involving MEK, Pl3K, kinase C and possibly cellular src. The TF-induced response did not in volve trans-activation of the EGF receptor kinase; but the converse (i .e., trans-activation of protease-activated receptor no. 1 (thrombin r eceptor) by the EGF receptor kinase) could not be ruled out.