TYROSINE KINASE INHIBITORS AND THE CONTRACTILE ACTION OF EPIDERMAL GROWTH-FACTOR - UROGASTRONE AND OTHER AGONISTS IN GASTRIC SMOOTH-MUSCLE

We examined the effects of the tyrosine kinase (TK) inhibitors, genistein, and tyrphostin (RG-50864) on the contractile action of epidermal growth factor - urogastrone (EGF-URO), transforming growth factor-alpha (TGF-alpha), and other agonists in two smooth muscle bioassay systems (guinea pig gastric longitudinal muscle, LM, and circular muscle, CM). We also studied the inhibition by tyrphostin of EGF-URO stimulated protein phosphorylation in identical smooth muscle strips. The selective inhibition by genistein and tyrphostin of EGF-URO and TGF-alpha induced contraction, but not of carbachol- and bradykinin-mediated contraction, occurred at much lower concentrations (genistein, < 7.4-mu-M (2-mu-g/mL); tyrphostin, < 20-mu-M (4-mu-g/mL) than those used in previously published studies with these TK inhibitors. In LM tissue, the IC50 values were for genistein 1.1 +/- 0.1-mu-M (0.30-mu-g/mL; mean +/- SEM) and 3.6 +/- 0.5-mu-M) (0.74-mu-g/mL) for tyrphostin, yielding a molar potency ratio (GS:TP) of 1:3 in the longitudinal preparation. In CM tissue, the IC50 values were 3.0 +/- 0.3-mu-M (0.81-mu-g/mL) for genistein and 2.4 +/- 0.2-mu-M (0.49-mu-g/mL) for tyrphostin, yielding a molar potency ratio (GS:TP) of 1.0:0.8 in the circular strips. The inhibition by genistein and tryphostin of EGF-URO and TGF-alpha mediated contraction was rapid (beginning within minutes) and was reversible upon washing the preparations free from the enzyme inhibitors. In intact tissue strips studied under bioassay conditions, tyrphostin (40-mu-M) also blocked EGF-URO triggered phosphorylation of substrates detected on Western blots using monoclonal antiphosphotyrosine antibodies. In contrast with the results with EGF-URO, genistein at 7.4-mu-M (2-mu-g/mL) and tyrphostin at 20-mu-M (4-mu-g/mL) had no effect on either bradykinin (1-mu-M) or carbachol (1-mu-M) stimulated contraction in both LM and CM preparations. However, the contractile action of prostaglandin F(2-alpha) (1-mu-M) was partially inhibited by genistein (7.4-mu-M), up to 10 +/- 5% in the LM preparation and 35 +/- 3% in the CM preparation (p < 0.05, LM vs. CM preparations) but not by tyrphostin (20-mu-M). Additionally, at the same concentrations, these tyrosine kinase inhibitors completely blocked angiotensin II mediated contraction in the LM preparation and partially inhibited contraction (43 +/- 3%, mean +/- SEM) in the CM preparation. The data suggest that tyrosine kinase activity plays an important role in EGF-URO induced contraction of gastric smooth muscle. Additionally, our results suggest that tyrosine kinase activity may also play a role in the signal transduction pathways by which smooth muscle contraction is induced by agonists such as angiotensin II and prostaglandin F(2-alpha), which are known to act via a G-protein receptor coupled pathway.