Elsevier

European Journal of Pharmacology

Volume 811, 15 September 2017, Pages 129-133
European Journal of Pharmacology

Pulmonary, gastrointestinal and urogenital pharmacology
Relaxation of isolated guinea-pig trachea by apigenin, a constituent of celery, via inhibition of phosphodiesterase

https://doi.org/10.1016/j.ejphar.2017.06.014Get rights and content

Abstract

Apigenin, was reported to have vasodilatory effects by inhibiting Ca2+ influx through both voltage- and receptor-operated calcium channels, but not by inhibiting cAMP- or cGMP-phosphodiesterases (PDEs) in rat thoracic aorta. However, apigenin was reported to inhibit PDE1, 2 and 3 in guinea-pig lung and heart. The aim of this study was to clarify that guinea-pig tracheal relaxation by apigenin whether via PDE inhibition.

We isometrically recorded the tension of isolated guinea-pig tracheal segments on a polygraph. Antagonistic effects of apigenin against cumulative contractile agents or Ca2+ induced contractions of the trachealis in normal or isotonic high-K+, Ca2+-free Krebs solution, respectively. Effects of apigenin (15 and 30 μM) on the cumulative forskolin- and nitroprusside-induced relaxations to histamine (30 μM)-induced precontraction were performed. The inhibitory effects of 30–300 μM apigenin and 3-isobutyl-1-methylxanthine (IBMX, positive control) on the cAMP- and cGMP-PDEs were determined.

Apigenin concentration-dependently but non-competitively inhibited cumulative histamine-, carbachol- or Ca2+-induced contractions in normal or in the depolarized (K+, 60 mM) trachealis, suggesting that Ca2+ influx through voltage-dependent calcium channels is inhibited. However, apigenin (15–30 μM) parallel leftward shifted the concentration-response curves of forskolin and nitroprusside, and significantly increased the pD2 values of these two cyclase activators. Both apigenin and IBMX, a reference drug, concentration (10–300 μM)-dependently and significantly, but non-selectively inhibited the activities of cAMP- and cGMP-PDEs in the trachealis. In conclusion, the relaxant effect of apigenin may be due to inhibition of both enzyme activities and reduction of intracellular Ca2+ by inhibiting Ca2+ influx in the trachealis.

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.

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