Pulmonary, gastrointestinal and urogenital pharmacologyRelaxation of isolated guinea-pig trachea by apigenin, a constituent of celery, via inhibition of phosphodiesterase
Introduction
Phosphodiesterases (PDEs) are classified according to their primary protein and complementary DNA sequences, co-factors, substrate specificities, and pharmacological roles. It is now known that PDEs comprise at least 11 distinct enzyme families that hydrolyze adenosine 3′,5′ cyclic monophosphate (cAMP) and/or guanosine 3′,5′ cyclic monophosphate (cGMP) (Lee et al., 2002). Thus PDEs are roughly classified to cAMP- and cGMP-PDEs. cAMP and cGMP are synthesized from ATP and GTP, when adenylate cyclase and guanylate cyclase are activated, respectively. If cAMP- or cGMP-PDEs are inhibited, the intracellular content of cAMP or cGMP is enhanced and subsequently activates cAMP- or cGMP-dependent protein kinase which may phosphorylate and inhibit myosin light-chain kinase, thus inhibiting contractions (Westfall et al., 1998).
Flavonoids at least divide into five classes (flavones, flavonols, flavanones, isoflavones, and chalcones). We previously reported that flavones, similar to isoflavones, are the most potent among these classes in guinea-pig tracheal relaxation (Ko et al., 2003). Apigenin, a member of flavones and also a constituent of Apium graveolens L. (Apiaceae), was reported to have vasodilatory effects by inhibiting Ca2+ influx through both voltage- and receptor-operated calcium channels, but not by enhancing cAMP or cGMP in rat thoracic aorta (Ko et al., 1991, Ajay et al., 2003). Their results suggest that the vasodilating effects of apigenin were unrelated to inhibition of PDEs in rat thoracic aorta. However, we reported that apigenin inhibited PDE1 (calcium/calmodulin-dependent), PDE2 (cGMP-stimulated) and PDE3 (cGMP-inhibited) of guinea-pig lung and heart with the IC50 values of 25.4, 16.7 and 10.5 µM, respectively (Ko et al., 2004). The inconsistency between our result and theirs may be due to tissue difference. The aim of this study was to clarify guinea-pig tracheal relaxant effects of apigenin whether via PDE inhibition.
Section snippets
Reagents and drugs
Apigenin (4′,5,7-trihydroxyflavone, molecular weight 270.24), aminophylline, calmodulin, cAMP, carbachol, α-chymotrypsin, cGMP, Crotalus atrox snake venom, 2′,5′-dideoxyadenosine, dl-dithiothreitol, Dowex resin, forskolin, glibenclamide, histamine, indomethacin, 3-isobutyl-1-methylxanthine (IBMX), methylene blue, nifedipine, Nω-nitro-L-arginine (L-NNA), nitroprusside, propranolol, sodium ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and Tris-HCl were purchased from
Non-competitive antagonism to contractile agents
Apigenin concentration-dependently inhibited the log concentration-response curves of cumulative histamine (Fig. 1A), carbachol (Fig. 1B), and KCl (Fig. 1C) in a non-competitive manner. The pD2′ values were 4.56 ± 0.09 (n = 10), 3.06 ± 0.13 (n = 6), and 3.80 ± 0.01 (n = 5), respectively, which significantly differed from each other (one-way ANOVA and then determined by Dunnett's test).
Isotonic high-K+-depolarized trachealis
Apigenin (25 and 100 μM) also concentration-dependently inhibited the log concentration-response curves of
Discussion
The log concentration-relaxing response curve of apigenin to the histamine (30 μM)-induced precontraction was not influnced by epithelium removal or propranolol (1 μM), a non-selective β-adrenoceptor blocker, suggesting that its relaxant effect is unrelated to the epithelium or activation of β-adrenoceptor. Neither 2′,5′-dideoxyadenosine, an adenylate cyclase inhibitor (Sabouni et al., 1991), nor methylene blue, a soluble guanylate cyclase inhibitor (Gruetter et al., 1981), influenced the log
Conclusions
Thus, the relaxant effects of apigenin may be due to inhibition of both cAMP- and cGMP-PDE activities and subsequent reduction of [Ca2+]i by inhibiting Ca2+ influx in the trachealis.
Authors’ contributions
WCK conceived and designed the study. JLC performed the experiments and analyzed the data. JLC and WCK wrote the manuscript. All the authors read and approved the final manuscript.
Conflict of interests
The authors declare that there is no conflict of interest regarding the publication of this paper.
Acknowledgments
We gratefully acknowledge that this work was supported by a grant (NSC 87–2314-B-038–039) from the Ministry of Science and Technology, Taipei, Taiwan.
References (18)
- et al.
Effects of flavonoids on vascular smooth muscle of the isolated rat thoracic aorta
Life Sci.
(2003) - et al.
Muscarinic acetylcholine receptor subtypes in smooth muscle
Trends Pharmacol. Sci.
(1994) - et al.
Vasodilatory action mechanisms of apigenin isolated from Apium graveolens in rat thoracic aorta
Biochim. Biophys. Acta
(1991) - et al.
Inhibitory effects of flavonoids on phosphodiesterase isozymes from guinea pig and their structure-activity relationships
Biochem. Pharmacol.
(2004) - et al.
Crystal structure of phosphodiesterase 4D and inhibitor complex (1)
FEBS Lett.
(2002) - et al.
pDx, pAx and pD'x values in the analysis of pharmacodynamics
Arch. Int. Pharm. Ther.
(1957) - et al.
Methylene blue inhibits coronary arterial relaxation and guanylate cyclase activation by nitroglycerin, sodium nitrite, and amyl nitrite
Can. J. Physiol. Pharmacol.
(1981) The influence of epithelium on the responsiveness of guinea-pig isolated trachea
Br. J. Pharmacol.
(1986)- et al.
N-omega-nitro-L-arginine: a potent inhibitor of endothelium-derived relaxing factor formation
Eur. J. Pharmacol.
(1990)