Effects of genistein on rat ileum smooth muscle contraction and histamine release from rat peritoneal mast cells

Abstract

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,...

Mode of action of histone deacetylase inhibitors on mast cell histamine release and colon muscle contraction

“…Methods for study of muscle contraction and histamine release from rat peritoneal mast cells (RPMC) were as previously described [6]. For antigen challenge, guinea-pigs were sensitised with egg albumin in an aluminium hydroxide suspension.…”

Pharmacological actions of a new synthetic cyclodepsipeptide, the A83586C-citropeptin hybrid, on complement C5a and G-Proteins

“…E. coli recombinant human C5a, AAPH (2,2'-azobis(2-amidinopropane)), benzalkonium choride (B-Cl), H 2 O 2 , 2-deoxyglucose and antimycin-A were from Sigma-Aldrich, and the 65-87 C-terminal human C5a peptide was from Bachem UK. Experiments on guinea-pig tracheal rings (GPTR), rat ileum (RI) smooth muscle and rat peritoneal mast cells (RPMC), were carried out as previously described [5][6][7] and according to UK legal regulations.…”

Cross-interaction between PAR-activation and tyrosine kinase in the modulation of rat ileum smooth muscle contraction

“…The materials and methods used were as previously described [3]. Briefl y, rat ileum was incubated with Krebs solution at 37 °C.…”

“…Serine proteases (proteinase-activated receptor, PAR, agonists), protein tyrosine kinases and protein phosphatases may interact to regulate smooth muscle activity [1,2]. We have studied the effect of N-tosyl-L-phenylalanine chloromethylketone (TPCK, a-chymotrypsin inhibitor), genistein (GEN, tyrosine kinase inhibitor) and cyclosporin A (CSA, type 2B protein phosphatase inhibitor) on the relaxation of sodium fl uoride (NaF, a non-specifi c G-protein activator)-and carbachol (CARB, a muscarinic receptor agonist)-induced rat ileum smooth muscle contraction (RISMC) by 5'N-ethylcarboxamido adenosine (NECA, adenosine A 2 agonist), a-chymotrypsin (a-CHY, PAR-2 agonist), GEN and isoprenaline (ISO, b 2 -agonist).…”

“…We have studied the effect of N-tosyl-L-phenylalanine chloromethylketone (TPCK, a-chymotrypsin inhibitor), genistein (GEN, tyrosine kinase inhibitor) and cyclosporin A (CSA, type 2B protein phosphatase inhibitor) on the relaxation of sodium fl uoride (NaF, a non-specifi c G-protein activator)-and carbachol (CARB, a muscarinic receptor agonist)-induced rat ileum smooth muscle contraction (RISMC) by 5'N-ethylcarboxamido adenosine (NECA, adenosine A 2 agonist), a-chymotrypsin (a-CHY, PAR-2 agonist), GEN and isoprenaline (ISO, b 2 -agonist). In addition, the effect of aminoguanidine (AMG, nitric oxide synthase inhibitor) was also investigated [2].…”