Relaxation of Isolated Guinea Pig Trachea by Genistein via Inhibition of Phosphodiesterase

Abstract

We investigated the mechanisms of the relaxant action of genistein, an isoflavone, phytoestrogen and non-specific protein tyrosine kinase inhibitor. Changes in tension of guinea pig tracheal segments were isometrically recorded on a polygraph. Genistein concentration-dependently relaxed histamine (30 μM)-, carbachol (0.2 μM)-, KCl (30 mM)- and leukotriene D₄ (10 nM)-induced precontractions and inhibited cumulative histamine- and carbachol-induced contractions in a non-competitive manner. Genistein also concentration-dependently and non-competitively inhibited the cumulative, Ca²⁺-induced contractions in the depolarized (K⁺, 60 mM) trachealis. The remaining nifedipine (10 μM)-induced tension of the histamine (30 μM)-induced precontraction was further relaxed by genistein, suggesting that regardless of whether voltage-dependent calcium channels are blocked genistein may have other mechanisms of relaxant action. These other mechanisms of the relaxant effect of genistein appeared to be epithelium-independent and were not affected by the presence of propranolol (1 μM), 2′,5′-dideoxyadenosine (10 μM), methylene blue (25 μM), glibenclamide (10 μM), N ^ω-nitro-L-arginine (20 μM) or α-chymotrypsin (1 U/mL), suggesting that the mechanisms are unrelated to activation of the β-adrenoceptor, of adenylate cyclase, of guanylate cyclase, of adenosine triphosphate-sensitive potassium channel opening, of nitric oxide formation or of neuropeptide release, respectively. However, genistein (17.5 - 35 μM) produced parallel, leftward shifts in the concentration-response curves of forskolin and nitroprusside and significantly increased the pD₂ values of these two agonists. Both genistein and 3-isobutyl-1-methylxanthine at various concentrations (10 - 300 μM) concentration-dependently and significantly inhibited cAMP- and cGMP-phosphodiesterase (PDE) activities of the trachealis. The -log IC₅₀ values of genistein were estimated to be 4.28 and 4.17, respectively. The above results reveal that the mechanisms of the relaxant action of genistein may be due to its non-selective inhibition of both PDE activities.

Abbreviations

IBMX:3-ixobutyl-1-methylxanthine

VDCCs:voltage-dependent calcium channels

cAMP:adenosine 3′,5′-cyclic monophosphate

cGMP:guanosine 3′,5′-cyclic monophosphate

ATP:adenosine triphosphate

PDE:phosphodiesterase

LTD₄:leukotriene D₄

L-NNA:N^ω-nitro-L-arginine

DMSO:dimethyl sulfoxide

EGTA:N,N,N′,N′-tetraacetic acid

ANOVA:analysis of variance

Key words

Genistein - isoflavone - phosphodiesterase inhibitor - guinea pig tracheal relaxation - cyclic AMP-phosphodiesterase - cyclic GMP-phosphodiesterase

References

• 1 Formica J V, Regelson W. Review of the biology of quercetin and related bioflavonoids.  Food Chem Toxicol. 1995;  33 1061-80.
• 2 Fotsis T, Pepper M S, Aktas E, Breit S, Rasku S, Adlercreutz H. et al . Flavonoids, dietary-derived inhibitors of cell proliferation and in vitro angiogenesis.  Cancer Res. 1997;  57 2916-21.
• 3 Wang H K, Xia Y, Yang Z Y, Natschke S L, Lee K H. Recent advances in the discovery and development of flavonoids and their analogues as antitumor and anti-HIV agents.  Adv Exp Med Biol. 1998;  439 191-225.
• 4 Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N. et al . Genistein, a specific inhibitor of tyrosine-specific protein kinases.  J Biol Chem. 1987;  262 5592-5.
• 5 Janssen L J, Lu-Chao H, Netherton S. Responsiveness of canine bronchial vasculature to excitatory stimuli and to cooling.  Am J Physiol Lung Cell Mol Physiol. 2001;  280 L930-7.
• 6 Janssen L J, Wattie J, Lu-Chao H, Tazzeo T. Muscarinic excitation-contraction coupling mechanisms in tracheal and bronchial smooth muscles.  J Appl Physiol. 2001;  91 1142-51.
• 7 Tsang F, Fred Wong W S. Inhibitors of tyrosine kinase signaling cascade attenuated antigen challenge of guinea-pig airways in vitro .  Am J Respir Crit Care Med. 2000;  162 126-33.
• 8 Stringfield T M, Morimoto B H. Modulation of cyclic AMP levels in a clonal neural cell line by inhibitors of tyrosine phosphorylation.  Biochem Pharmacol. 1997;  53 1271-8.
• 9 Ueki H, Mitsugi S, Kawashima Y, Motoyashiki T, Morita T. Orthovanadate stimulates cyclic guanosine monophosphate-inhibited cyclic adenosine monophosphate phosphodiesterase activity in isolated rat fat pads through activation of particulate myelin basic protein kinase by protein tyrosine kinase.  Endocrinology. 1997;  138 2784-9.
• 10 O’Connell J C, McCallum J F, McPhee I, Wakefield J, Houslay E S, Wishart W. et al . The SH3 domain of Src tyrosyl protein kinase interacts with the N-terminal splice region of the PDE4A cAMP-specific phosphodiesterase RPDE-6 (RNPDE4A5).  Biochem J. 1996;  318 255-61.
• 11 Nichols M R, Morimoto B H. Tyrosine kinase-independent inhibition of cyclic-AMP phosphodiesterase by genistein and tyrphostin 51.  Arch Biochem Biophys. 1999;  366 224-30.
• 12 Holroyde M C. The influence of epithelium on the responsiveness of guinea-pig isolated trachea.  Br J Pharmacol. 1986;  87 501-7.
• 13 Cook S J, Archer K, Martin A, Buchheit K H, Fozard J R, Muller T. et al . Further analysis of the mechanisms underlying the tracheal relaxant action of SCA40.  Br J Pharmacol. 1995;  114 143-51.
• 14 Ariens E J, van Rosssum J M. pD_x, pA_x and pD′_x values in the analysis of pharmacodynamics.  Arch Int Pharmacodyn Ther. 1957;  110 275-99.
• 15 Tsien R W. Calcium channels in excitable cell membranes.  Annu Rev Physiol. 1983;  45 341-58.
• 16 Ko W C, Kuo S W, Sheu J R, Lin C H, Tzeng S H, Chen C M. Relaxant action mechanisms of quercetin 3,3′,4′,5,7-pentamethyl ether in isolated guinea-pig trachea.  New Taipei J Med. 1999;  1 98-106.
• 17 Sabouni M H, Cushing D J, Makujina S R, Mustafa S J. Inhibition of adenylate cyclase attenuates adenosine receptor-mediated relaxation in coronary artery.  J Pharmacol Exp Ther. 1991;  259 508-12.
• 18 Gruetter C A, Kadowitz P J, Ignarro L J. Methylene blue inhibits coronary arterial relaxation and guanylate cyclase activation by nitroglycerin, sodium nitrite, and amyl nitrite.  Can J Physiol Pharmacol. 1981;  59 150-6.
• 19 Murray M A, Boyle J P, Small R C. Cromakalim-induced relaxation of guinea-pig isolated trachealis: antagonism by glibenclamide and by phentolamine.  Br J Pharmacol. 1989;  98 865-74.
• 20 Ishii K, Chang B, Kerwin JF J r, Huang Z J, Murad F. N-omega-nitro-L-arginine: a potent inhibitor of endothelium-derived relaxing factor formation.  Eur J Pharmacol. 1990;  176 219-23.
• 21 Urakawa N, Holland W C. Ca⁴⁵ uptake and tissue calcium in K-induced phasic and tonic contraction in taenia coli.  Am J Physiol. 1964;  207 873-6.
• 22 Goodman F R, Weiss G B, Karaki H, Nakagawa H. Differential calcium movements induced by agonists in guinea pig tracheal muscle.  Eur J Pharmacol. 1987;  133 111-7.
• 23 Eglen R M, Reddy H, Watson N, Challiss R A. Muscarinic acetylcholine receptor subtypes in smooth muscle.  Trends Pharmacol Sci. 1994;  15 114-9.
• 24 Ko W C, Lei C B, Lin Y L, Chen C F. Mechanisms of relaxant action of S-petasin and S-isopetasin, sesquiterpenes of Petasites formosanus, in isolated guinea pig trachea.  Planta Med. 2001;  67 224-9.
• 25 Seamon K B, Daly J W, Metzger H, de Souza N J, Reden J. Structure-activity relationships for activation of adenylate cyclase by the diterpene forskolin and its derivatives.  J Med Chem. 1983;  26 436-9.
• 26 Schultz K, Schultz K, Schultz G. Sodium nitroprusside and other smooth muscle-relaxants increase cyclic GMP levels in rat ductus deferens.  Nature. 1977;  265 750-1.
• 27 Westfall D P, Gerthoffer W T, Webb R C. Vasodilators and nitric oxide synthase. In: Human pharmacology molecular to clinical. Brody TM, Larner J, Minneman KP, editors. St.  Louis:. Mosby;  1998 239-47.