IntroductionTyrosine phosphorylation of proteins plays a major role in a variety of cellular signalling pathways. Genistein (a tyrosine kinase inhibitor) is an isoflavone phytoestrogen (found in soy). Amongst its many actions, genistein inhibits the following responses: antigen-induced smooth muscle contraction (possibly by inhibiting histamine release (HR) from mast cells) [1], inflammation in mice (inhibiting delayed type hypersensitivity and collagen-induced arthritis) [2], NO synthesis in rat aorta [3] and GTP-induced contraction as well as Ca 2+ fluxes in rat mesenteric artery [4], suggesting that it might influence G-protein activity. We have now studied: (1) effect of genistein on rat ileum smooth muscle contraction (ISMC) induced by sodium fluoride (NaF, a non-specific Gprotein activator) and carbachol, (2) effect of aminoguanidine (AMG, a NO synthesis inhibitor), sodium nitroprusside (SNP, a NO donor), N-tosyl-L-phenylalanine chloromethyl ketone (TPCK, a-chymotrypsin inhibitor), disodium cromoglycate (DSCG, mast cell 'stabilizer') and calcium ionophore A23187 on genistein action in ISMC, and (3) effect of genistein on NaF-induced HR from rat peritoneal mast cells (RPMC).
Materials and methodsMale Sprague-Dawley rats (250-330 g) were used in all experiments. The materials and methods used were as previously described [5], and according to local ethical committee and Home Office regulations.
Ileal smooth muscle contractionNaF or carbachol, at stimulatory concentrations, were added to one ileum preparation, once before and once after the 15 min incubation with a given concentration of genistein. DSCG, SNP and TPCK were administered together with genistein for 15 min, whereas A23187 or AMG were added 15 min before genistein. The results were expressed as the percentage reduction of stimulant-induced contraction.Washed mixed RPMC were incubated in HEPES-buffered Tyrode solution (pH 7.4) at 37°C with genistein at designated concentrations (5-100 mM) for 10 min, followed by stimulation with NaF (5-30 mM) for a further 30 min. The rest of the procedure was as described previously [5].
MaterialsGeneral chemicals were from BDH/Merck Ltd., Poole, UK. Biochemicals were from Sigma (Poole, UK) except for DSCG, which was a gift from Fisons Pharmaceuticals. They were dissolved in saline (carbachol, histamine, DSCG, SNP and AMG), in distilled water (NaF and TPCK) or in 0.15% dimethylsulphoxide (A23187).
Results and discussionSubmaximal concentrations of NaF (10 mM) and carbachol (0.5 mM) induced a contraction greater than 50% above basal level. Genistein (0.1-100 mM) produced a dose-related relaxation of NaF-and carbachol-induced ISMC with IC 50 s of 1 mM and 100 mM, respectively. Thus, genistein was much less effective against carbachol-induced ISMC.A23187 (1 mM) and SNP as well as AMG (both at 100 mM) had a mild inhibitory effect on the action of genistein (1 mM) on NaF-induced ISMC. DSCG (100 mM) markedly and significantly (p < 0.01, Student's t-test) reduced the relaxant effect of genistein (1 mM) on NaFinduced ISMC,...
