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

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

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