Tyrosine kinase inhibitors and the contractile action of epidermal growth factor – urogastrone and other agonists in gastric smooth muscle

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Abstract
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-α (TGF-α), 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-α induced contraction, but not of carbachol- and bradykinin-mediated contraction, occurred at much lower concentrations (genistein, 50 values were for genistein 1.1 ± 0.1 μM (0.30 μg/mL; mean ± SEM) and 3.6 ± 0.5 μM (0.74 μg/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 μM (0.81 μg/mL) for genistein and 2.4 ± 0.2 μM (0.49 μg/mL) for tyrphostin, yielding a molar potency ratio (GS:TP) of 1.0:0.8 in the circular strips. The inhibition by genistein and tyrphostin of EGF–URO and TGF-α 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 μM) 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 μM (2 μg/mL) and tyrphostin at 20 μM (4 μg/mL) had no effect on either bradykinin (1 μM) or carbachol (1 μM) stimulated contraction in both LM and CM preparations. However, the contractile action of prostaglandin F2α (1 μM) was partially inhibited by genistein (7.4 μM), 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 μM). 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 F2α which are known to act via a G-protein receptor coupled pathway.Key words: smooth muscle, tyrosine kinase, growth factor, angiotensin.

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