Wun-Chang Ko

Luteolin, a non-selective competitive inhibitor of phosphodiesterases 1–5, displaced [3H]-rolipram from high-affinity rolipram binding sites and reversed xylazine/ketamine-induced anesthesia

The aim of the present study was to investigate the mode of action of luteolin on phisphodiesterase (PDE) 1-5, and the possible adverse effects, such as nausea, vomiting, and gastric hypersecretion, determined by replacing [(3)H]-rolipram binding and reversing xylazine/ketamine-induced anesthesia. The reversing effect was reported to occur through a presynaptic alpha(2)-adrenoceptor inhibition and trigger vomiting in ferrets. In contrast, clonidine, an alpha(2)-adrenoceptor agonist, prevented emesis induced by PDE4 inhibitors in ferrets. According to the Lineweaver-Burk analysis, luteolin (3-30 microM) competitively inhibited PDE1-5 activities, with K(i) values of 15.0, 6.4, 13.9, 11.1, and 9.5 microM, respectively, which did not significantly differ from each other. The equilibrium dissociation constant (K(d)) and maximal density (B(max)) for [(3)H]-rolipram binding at high-affinity rolipram binding sites of guinea pig brain cell membranes were 10.1 nM and 3.7 p mol/g of tissue, respectively. The EC(50) (PDE4(H)) values of luteolin and Ro 20-1724, a selective PDE4 inhibitor, for displacing 2 nM [(3)H]-rolipram binding were 11.2 microM and 45.6 nM, respectively. The therapeutic (PDE4(H)/PDE4(L)) ratios of luteolin and Ro 20-1724 were calculated to be 0.6, and 0.004, respectively. Both luteolin (10-30 micromol/kg, s.c.) and Ro 20-1724 (0.1-1 micromol/kg, s.c.) significantly reversed the xylazine/ketamine-induced anesthesia in mice. Although luteolin non-selectively and competitively inhibited PDE1-5, only PDE4 inhibition contributed to a reversing effect. In conclusion, because of the low therapeutic (PDE4(H)/PDE4(L)) ratio of luteolin, the gastrointestinal adverse effects such as nausea, vomiting and gastric hypersecretion should be carefully monitored, whenever luteolin is used for treating allergies, asthma or chronic obstructive pulmonary disease.

Biochanin A, a Phytoestrogenic Isoflavone with Selective Inhibition of Phosphodiesterase 4, Suppresses Ovalbumin-Induced Airway Hyperresponsiveness

The present study investigated the potential of biochanin A, a phytoestrogenic isoflavone of red clover (Triflolium pratense), for use in treating asthma or chronic obstructive pulmonary disease (COPD). Biochanin A (100 μmol/kg, orally (p.o.)) significantly attenuated airway resistance (R L), enhanced pause (P enh), and increased lung dynamic compliance (C dyn) values induced by methacholine (MCh) in sensitized and challenged mice. It also significantly suppressed an increase in the number of total inflammatory cells, neutrophils, and eosinophils, and levels of cytokines, including interleukin (IL)-2, IL-4, IL-5, and tumor necrosis factor (TNF)-α in bronchoalveolar lavage fluid (BALF) of the mice. However, it did not influence interferon (IFN)-γ levels. Biochanin A (100 μmol/kg, p.o.) also significantly suppressed the total and ovalbumin (OVA)-specific immunoglobulin E (IgE) levels in the serum and BALF, and enhanced the total IgG2a level in the serum of these mice. The PDE4H/PDE4L value of biochanin A was calculated as >35. Biochanin A did not influence xylazine/ketamine-induced anesthesia. Biochanin A (10~30 μM) significantly reduced cumulative OVA (10~100 μg/mL)-induced contractions in the isolated guinea pig trachealis, suggesting that it inhibits degranulation of mast cells. In conclusion, red clover containing biochanin A has the potential for treating allergic asthma and COPD.

Hesperetin, a Selective Phosphodiesterase 4 Inhibitor, Effectively Suppresses Ovalbumin-Induced Airway Hyperresponsiveness without Influencing Xylazine/Ketamine-Induced Anesthesia

Hesperetin, a selective phosphodiesterase (PDE)4 inhibitor, is present in the traditional Chinese medicine, “Chen Pi.” Therefore, we were interested in investigating its effects on ovalbumin- (OVA-) induced airway hyperresponsiveness, and clarifying its rationale for ameliorating asthma and chronic obstructive pulmonary disease (COPD). Hesperetin was revealed to have a therapeutic (PDE4H/PDE4L) ratio of >11. Hesperetin (10 ~ 30 μmol/kg, intraperitoneally (i.p.)) dose-dependently and significantly attenuated the airway hyperresponsiveness induced by methacholine. It also significantly suppressed the increases in total inflammatory cells, macrophages, lymphocytes, neutrophils, and eosinophils, and levels of cytokines, including interleukin (IL)-2, IL-4, IL-5, interferon-γ, and tumor necrosis factor-α in bronchoalveolar lavage fluid (BALF). It dose-dependently and significantly suppressed total and OVA-specific immunoglobulin E levels in the BALF and serum. However, hesperetin did not influence xylazine/ketamine-induced anesthesia, suggesting that hesperetin has few or no emetic effects. In conclusion, the rationales for ameliorating allergic asthma and COPD by hesperetin are anti-inflammation, immunoregulation, and bronchodilation.

S-Petasin, the Main Sesquiterpene of Petasites formosanus, Inhibits Phosphodiesterase Activity and Suppresses Ovalbumin-Induced Airway Hyperresponsiveness

S-Petasin is the main sesquiterpene of Petasites formosanus, a traditional folk medicine used to treat hypertension, tumors and asthma in Taiwan. The aim of the present study was to investigate its inhibitory effects on phosphodiesterase (PDE) 1–5, and on ovalbumin (OVA)-induced airway hyperresponsiveness (AHR) in a murine model of allergic asthma. S-Petasin concentration-dependently inhibited PDE3 and PDE4 activities with 50% inhibitory concentrations (IC50) of 25.5, and 17.5 μM, respectively. According to the Lineweaver-Burk analysis, S-petasin competitively inhibited PDE3 and PDE4 activities with respective dissociation constants for inhibitor binding (K i) of 25.3 and 18.1 μM, respectively. Both IC50 and K i values for PDE3 were significantly greater than those for PDE4. S-Petasin (10–30 μmol/kg, administered subcutaneously (s.c.)) dose-dependently and significantly attenuated the enhanced pause (P enh) value induced by methacholine (MCh) in sensitized and challenged mice. It also significantly suppressed the increases in total inflammatory cells, lymphocytes, neutrophils, eosinophils and levels of cytokines, including interleukin (IL)-2, IL-4 and IL-5, tumor necrosis factor (TNF)-α and interferon (IFN)-γ in bronchoalveolar lavage fluid (BALF) of these mice. In addition, S-petasin (10–30 μmol/kg, s.c.) dose-dependently and significantly attenuated total and OVA-specific immunoglobulin E (IgE) levels in the serum and BALF, and enhanced the IgG2a level in serum of these mice. The PDE4H value of S-petasin was >300 μM; therefore, its PDE4H/PDE4L value was calculated to be >17. In conclusion, the present results for S-petasin at least partially explain why Petasites formosanus is used as a folk medicine to treat asthma in Taiwan.

Butylidenephthalide Blocks Potassium Channels and Enhances Basal Tension in Isolated Guinea-Pig Trachea

Butylidenephthalide (Bdph, 30~300 μM), a constituent of Ligusticum chuanxiong Hort., significantly enhanced tension in isolated guinea-pig trachea. In this study, we investigate the mechanism(s) of Bdph-induced contraction in the tissue. Isolated trachea was bathed in 5 mL of Krebs solution containing indomethacin (3 μM), and its tension changes were isometrically recorded. Cromakalim (3 μM), an ATP-dependent K+ channel opener, significantly antagonized the Bdph-induced enhancement of baseline tension. Bdph (300 μM) also significantly antagonized cromakalim-induced relaxation. Bdph (300 μM) did not significantly influence the antagonistic effects of glibenclamide (GBC, 1 μM) and tetraethylammonium (TEA, 8 mM) against the cromakalim-induced relaxation. However, Bdph (300 μM) and 4-aminopiridine (4-AP, 5 mM), a blocker of Kv1 family of K+ channels, in combination significantly rightward shifted the log concentration-relaxation curve of cromakalim. The antagonistic effect of the combination almost equals the sum of the individual effects of Bdph and 4-AP, suggesting that the antagonistic mechanism of Bdph may be similar to that of 4-AP. All calcium channel blockers influenced neither the baseline tension nor antagonistic effect of Bdph against cromakalim. In conclusion, Bdph may be similar to 4-AP, a blocker of Kv1 family of K+ channels, to enhance the baseline tension of guinea-pig trachea.

Genistein, a competitive PDE1-4 inhibitor, may bind on high-affinity rolipram binding sites of brain cell membranes and then induce gastrointestinal adverse effects.

The affinities of genistein on phosphodiesterase (PDE)1-4 and cause of gastrointestinal adverse effects of genistein remain unclear. Female BALB/c mice were actively sensitized by intraperitoneal injections of ovalbumin and challenged by aerosolized ovalbumin (1%). After secondary challenge, aerosolized methacholine (6.25-50mg/ml) induced increases of enhanced pause (P(enh)) values in conscious mice in a concentration-dependent manner. Genistein (30-100 micromol/kg, i.p.) markedly inhibited methacholine (12.5-50mg/ml)-induced increase of P(enh) value in the sensitized and challenged mice. In addition, genistein significantly reduced total inflammatory cells, macrophages, lymphocytes, neutrophils, and eosinophils in bronchoalveolar lavage fluid, with the exception that lymphocytes and neutrophils were not significantly inhibited by genistein at the lowest dose (10 micromol/kg). Genistein also markedly attenuated the release of cytokines, including interleukin (IL)-2, IL-4, IL-5, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha. Genistein competitively inhibited PDE1-4, with a K(i) value ranging from 4.3 to 13.7 microM. Genistein (3-300 microM) concentration-dependently displaced 2nM [(3)H]-rolipram bound on high-affinity rolipram binding sites of brain cell membranes. The therapeutic ratio of genistein was calculated to be 7.9. Genistein (100 micromol/kg, s.c.) significantly shortened xylazine/ketamine-induced anesthesia, suggesting that genistein administered at a higher dose may have gastrointestinal adverse effects. In conclusion, owing to the low therapeutic ratio of genistein, the gastrointestinal adverse effects may be induced via the binding of genistein on high-affinity rolipram binding sites of brain cell membranes, when it is used for a long term or at higher doses for treating allergies, asthma or chronic obstructive pulmonary disease.

Hesperidin-3′- O -methylether is more potent than hesperidin in phosphodiesterase inhibition and suppression of ovalbumin-induced airway hyperresponsiveness

Hesperidin is present in the traditional Chinese medicine, “Chen Pi,” and recently was reported to have anti-inflammatory effects. Therefore, we were interested in comparing the effects of hesperidin and hesperidin-3′-O-methylether on phosphodiesterase inhibition and airway hyperresponsiveness (AHR) in a murine model of asthma. In the present results, hesperidin-3′-O-methylether, but not hesperidin, at 30 μmol/kg (p.o.) significantly attenuated the enhanced pause (P enh) value, suppressed the increases in numbers of total inflammatory cells, macrophages, lymphocytes, neutrophils, and eosinophils, suppressed total and OVA-specific immunoglobulin (Ig)E levels in the serum and BALF, and enhanced the level of total IgG2a in the serum of sensitized and challenged mice, suggesting that hesperidin-3′-O-methylether is more potent than hesperidin in suppression of AHR and immunoregulation. The different potency between them may be due to their aglycons, because these two flavanone glycosides should be hydrolyzed by β-glucosidase after oral administration. Neither influenced xylazine/ketamine-induced anesthesia, suggesting that they may have few or no adverse effects, such as nausea, vomiting, and gastric hypersecretion. In conclusion, hesperidin-3′-O-methylether is more potent in phosphodiesterase inhibition and suppression of AHR and has higher therapeutic (PDE4H/PDE4L) ratio than hesperidin. Thus, hesperidin-3′-O-methylether may have more potential for use in treating allergic asthma and chronic obstructive pulmonary disease.

Relaxation of isolated guinea-pig trachea by apigenin, a constituent of celery, via inhibition of phosphodiesterase.

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 30μM) on the cumulative forskolin- and nitroprusside-induced relaxations to histamine (30μM)-induced precontraction were performed. The inhibitory effects of 30-300μM 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+, 60mM) trachealis, suggesting that Ca2+ influx through voltage-dependent calcium channels is inhibited. However, apigenin (15-30μM) 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 (10-300μM)-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.

Mechanisms of butylidenephthalide for twitch facilitation in electrically stimulated mouse vas deferens

Abstract Context: The rhizome of Ligusticum chuaxiong Hort. (Umbelliferae) has been used by Chinese for several thousand years. Its main constituent, butylidenephthalide (Bdph), was proved to be active in inhibiting rat uterine contractions induced by prostaglandin F2α and was reported to be a nonspecific antispamodic and a blocker of voltage-dependent Ca2+ channels (VDCCs). Objectives: The present study investigates the mechanisms of Bdph for twitch facilitation in ICR mouse vas deferens (MVD). Materials and methods: Electrical field stimulation (EFS, supramaximal voltage ranging from 60–90 V, 1 ms, 0.2 Hz) was applied to the isolated MVD in Krebs solution. Interactions between Bdph (50 µM) and calcium antagonist (verapamil, diltiazem or aspaminol) on the EFS-evoked twitch responses were determined. The number of experiments was 3–18. Results: Bdph (50 µM)-induced twitch facilitations from 100 to 391.9% were unrelated to activation of postjunctional cholinergic or adrenergic receptors. Verapamil and Bdph unabolished the twitch facilitation each other. Diltiazem unabolished the Bdph-induced twitch facilitation. In contrast, Bdph abolished those induced by diltiazem. Aspaminol at 20 μM abolished the Bdph-induced twitch facilitation. In contrast, Bdph abolished those induced by aspaminol. Tetraethylammonium and 4-aminopyridine, the K+ channel blockers, significantly augmented the Bdph-induced twitch facilitation. Discussion and conclusions: Bdph may bind to the different, more and same subtypes of VDCCs from verapamil, than diltiazem, and as aspaminol does on prejunctional membrane, respectively. Besides a blocker of VDCCs, Bdph may be a blocker of K+ channels on prejunctional membrane. Thus, Bdph depolarized the membrane and facilitated the cumulative Ca2+-induced twitch responses.