Hesperetin-5,7,3'-O-Trimethylether Dually Inhibits Phosphodiesterase 3/4 and Methacholine-Induced Airway Hyperresponsiveness in Sensitized and Challenged Mice.

INTRODUCTION: Hesperetin-5,7,3'-O-trimethylether (HTME), a synthetic liposoluble hesperetin, has been reported to be a dual phosphodiesterase (PDE)3/4 inhibitor. We investigated its inhibitory effects on methacholine (MCh)-induced airway hyperresponsiveness (AHR) and its potential for treating atypical asthma and COPD. METHODS: FlexiVent system was used to determine AHR in ovalbumin (OVA) sensitized and challenged mice. Determination of cytokines was performed by using mouse T helper (Th)1/Th2 cytokine CBA kits, and of total immunoglobulin (Ig)E and OVA-specific IgE using ELISA kits. The number of inflammatory cells was counted using a hemocytometer. Xylazine/ketamine-induced anesthesia was to assess nausea, vomiting, and gastric hypersecretion in these mice. RESULTS: HTME dually and competitively inhibited PDE3/4 activities in the Lineweaver-Burk analysis. HTME (30 and 100 µmol/kg) dose-dependently and significantly decreased the airway resistance (RL) and increased lung dynamic compliance (Cdyn) values induced by MCh. It significantly suppressed numbers of total inflammatory cells and neutrophils, and levels of cytokines in bronchoalveolar lavage fluid (BALF). HTME dose-dependently and significantly inhibited total and OVA-specific IgE levels in the BALF and serum. However, HTME did not influence xylazine/ketamine-induced anesthesia. CONCLUSION: HTME exerted anti-inflammatory and bronchodilator effects and may be useful in treating chronic obstructive pulmonary disease and allergic atypical asthma with no gastrointestinal side effects.

Butylidenephthalide facilitates contractions via nonspecific binding to receptors in isolated guinea-pig vas deferens.

Context: Butylidenephthalide (Bdph) has been reported to inhibit rat uterine contractions, but significantly potentiate the noradrenaline (NA)-induced contractions in guinea-pig vas deferens (GPVDs). Objective: The present study elucidates the binding specificity of Bdph in GPVD to potentiate contractions. Materials and methods: Electrical field stimulation (EFS, supramaximal voltage, 1 ms and 1 Hz) or exogenous NA (50 µM) was applied to the GPVD in Krebs or 1/10 Mg-Tyrode's solution, respectively. After the clonidine (10 nM)-induced twitch inhibition or the exogenous NA-induced contractions reached a constant, Bdph (50 µM) was added 2 min prior to the subsequent addition of NA (50 µM). Three experiments were performed. In the presence of Bdph (100 µM), the release of NA in the medium and remaining NA content in the tissues were determined after EFS-stimulation. Results: Bdph (100 µM) significantly antagonized the clonidine (10 nM)-induced twitch inhibition from 22.5 ± 2.1 to -11.4 ± 1.6% (n = 6) and dibutyryl-cAMP (300 µM) from 25.7 ± 3.2 to 7.9 ± 4.0% (n = 8). Bdph (100 µM) significantly increased the electrically stimulated release of NA from 393.0 ± 109.5 to 1000.0 ± 219.1 ng/g (n = 6). Bdph (50 µM) potentiated the exogenous NA (50 µM)-induced contractions from 3.0 ± 0.06 to 3.9 ± 0.06 g (n = 3), but after washout of Bdph, the response to NA gradually curtailed. Discussion and conclusions: Bdph action may be through the nonspecific binding of the butylidene group to prejunctional α2- and postjunctional α1-adrenoceptors to reversibly block K+ channels, and irreversibly block VDCCs on the smooth muscle cell membrane, respectively.

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

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.

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.

Hesperetin-5,7,3'-O-triacetate suppresses airway hyperresponsiveness in ovalbumin-sensitized and challenged mice without reversing xylazine/ketamine-induced anesthesia in normal mice.

BACKGROUND: We recently reported that hesperetin-5,7,3'-O-triacetate (HTA) dually inhibited phosphodiesterase (PDE)3/4 with a therapeutic ratio of 20.8. The application and development of PDE4 inhibitors for treating asthma or COPD are limited by their side effects, such as nausea, vomiting and gastric hypersecretion. PDE4 inhibitors were reported to reverse xylazine/ketamine-induced anesthesia in rats and triggered vomiting in ferrets. Thus the reversing effect of HTA on xylazine/ketamine-induced anesthesia in mice was studied to assess emetic effect of HTA. The aim of this study was to prove the therapeutic effect of HTA without vomiting effect at an effective dose for treating COPD. METHODS: Ten female BALB/c mice in each group were sensitized by ovalbumin (OVA) on days 0 and 14. On day 21, these mice were emphasized the sensitization by Freund's complete adjuvant. Mice were challenged by 1% OVA nebulization on days 28, 29, and 30. Airway hyperresponsiveness (AHR) was assessed on day 32 in each group, using the FlexiVent system to determine airway resistance (RL) and lung dynamic compliance (Cdyn) in anesthetized ovalbumin (OVA)-sensitized and challenged mice. Each group was orally administered HTA (10 ~ 100 µmol/kg), roflumilast (1 and 5 mg/kg) or vehicles (controls) 2 h before and 6 and 24 h after OVA provocation. For comparison, sham-treated mice were challenged with saline instead of 1% OVA. The ability to reverse xylazine/ketamine-induced anesthesia by HTA or roflumilast for 3 h was determined in normal mice. We used roflumilast, a selective PDE4 inhibitor and bronchodilator for severe COPD approved by the US Food and Drug Administration, as a reference drug. RESULTS: In the results, HTA (100 µmol/kg, p.o.) or roflumilast (5 mg/kg, p.o.) significantly suppressed all RL values of MCh at 0.78 ~ 25 mg/mL and enhanced Cdyn values of MCh at 3.125 ~ 25 mg/mL compared to OVA-sensitized and -challenged control mice. Orally administered 1, 3 or 10 mg/kg roflumilast, but not 30 or 100 µmol/kg HTA, significantly reversed xylazine/ketamine-induced anesthesia. CONCLUSIONS: In contrast to roflumilast, HTA may ameliorate COPD but induce few side effects of nausea, vomiting and gastric hypersecretion at an effective dose for treating COPD, because HTA did not reverse xylazine/ketamine-induced anesthesia in mice.

Stereoselectivity of butylidenephthalide on non-adrenergic prejunctional voltage-dependent Ca(2+) channels in prostatic portion of rat vas deferens.

The naturally occurring and synthetic butylinenephthalide (Bdph) has two geometric isomers. Z- and E-Bdph were reported to have geometric stereoselectivity for voltage-dependent calcium channels (VDCCs) in guinea-pig ileum. The aim of this study was to investigate whether the binding of Z- and E-Bdph on prejunctional VDCCs of rat vas deferens (RVD) is stereoselective. The twitch responses to electrical field stimulation (EFS, supramaximal voltage, 1 ms, 0.2Hz) were recorded on a polygraph. Z- and E-Bdph concentration-dependently inhibited the twitch responses to EFS in full tissue, prostatic portion and epididymal portion of RVD. The pIC50 value of Z-Bdph was greater than that of E-Bdph in the electrically stimulated prostatic portion of RVD, suggesting that the binding of Bdph on the non-adrenergic prejunctional VDCCs of cell membrane is stereoselective. In the prostatic portion, exogenous Ca(2+) only partially reversed the twitch inhibition by Z-Bdph, but effectively reversed those by Ca(2+) channel blockers, such as verapamil, diltiazem and aspaminol, suggesting that the action mechanisms may be different from those of Ca(2+) channel blockers. K(+) channel blockers, such as tetraethylammonium (TEA) and 4-aminopyridine (4-AP), may prolong duration of action potential to allow greater Ca(2+) entry and induced more release of transmitters. Therefore both blockers via their prejunctional actions reversed the twitch inhibition induced by Z-Bdph in all preparations of RVD by a non-specific antagonism.

Interaction Between Daidzein and Hesperetin on Antispasmodic Action in Isolated Sensitized and Non-sensitized Guinea-Pig Tracheas.

In traditional Chinese medicine (TCM), a combination of kudzu and Chen-Pi is frequently prescribed for relieving colds, fever, bronchitis, and cough. It contains daidzein and hesperetin, selective inhibitors of family 3 (PDE3), and 4 (PDE4) of phosphodiesterases (PDEs), respectively. In passively sensitized human airways, allergen-induced contraction was reported to be inhibited only by the simultaneous inhibition of PDE3 and PDE4, but not by single inhibition of either isozyme. Therefore, we are interested in investigating the interaction between daidzein and hesperetin on their antispasmodic effects in the isolated sensitized and non-sensitized guinea-pig tracheas, to clarify the difference between these two tissues, because effects of TCM prescription on patients with or without allergic asthma are often different. Guinea-pigs were sensitized by subcutaneous injection of ovalbumin (OVA) into legs. After sensitization, the baseline and cumulative OVA-induced contractions of the sensitized trachea were isometrically recorded on a polygraph. In the same way, the histamine (30 µM)-induced tonic contraction of non-sensitized guinea-pig trachea was recorded. The isobole method was used to analyze the antagonism and synergism between daidzein and hesperetin. The isoboles showed antagonism between daidzein and hesperetin on baseline relaxant effect and OVA (100 µg/ml)-induced contraction in the sensitized guinea-pig trachea. In contrast, the isobole showed synergism between daidzein and hesperetin on the relaxant effect of histamine-induced tonic contraction in non-sensitized guinea-pig trachea. These results suggest that the combination of kudzu and Chen-Pi for relieving colds, fever, bronchitis and cough is effective in patients without, but might show little effect in patients with allergic asthma.

Lack of effect of Z-butylidenephthalide on presynaptic N-type Ca²âº channels in isolated guinea-pig ileum.

Z-Butylidenephthalide (Bdph) was reported to more potently inhibit electrically induced twitch responses than acetylcholine-induced tonic contraction in isolated guinea-pig ileum (GPI). The aim of the present study was to investigate the inhibitory effects of Z-Bdph on Ca2+ and K+ channels on GPI. In Locke-Ringer's solution, both responses were isometrically recorded on a polygraph. Incubation of ω-conotoxin MVIIC, but not Z-Bdph, in the electrically stimulated GPI prior to adding ω-conotoxin GVIA, an irreversible blocker of N-type voltage-dependent Ca2+ channels (VDCCs), protected the binding sites and resulted in the twitch responses reversible by washing, suggesting that Z-Bdph did not bind to the N-type VDCCs. Interestingly, we found Z-Bdph concentration dependently delayed the onsets of K+-induced twitch responses, suggesting that Z-Bdph may be a blocker of K+ channels to interfere extracellular K+ across through the pre-junctional membrane of nerve ending in K+-free medium. Z-Bdph similar to nifedipine non-competitively inhibited cumulative ACh-induced phasic contractions, suggesting that Z-Bdph may bind to L-type of inositol-1,4,5-trisphosphate (IP3)-sensitive Ca2+ channels on the endoplasmic reticulum (ER) membrane. In the presence of verapamil, a L-type Ca2+ channel blocker or Z-Bdph, the twitch inhibitions by either were effectively reversed by exogenous Ca2+, suggesting that they may freely pass through pre-junctional N-type, but not L-type which was blocked at least a part by either, of VDDCs open when each electrical coaxial stimulation (ECS) into intracellular space of cholinergic nerve terminal and trigger release of transmitters. In conclusion, results confirm that Z-Bdph more potently inhibits ECS-induced twitch responses than ACh-induced PCs in GPI and suggest that this effect is not mediated by interaction with presynaptic N-type VDCCs.

Butylidenephthalide antagonizes cromakalim-induced systolic pressure reduction in conscious normotensive rats.

BACKGROUND: Butylidenephthalide (Bdph), a main constituent of Ligusticum chuanxiong Hort., was reported to have selective antianginal effect without changing blood pressure in conscious rat. Recently, we have observed that Bdph antagonized cromakalim, an ATP-dependent K(+) channel opener, in guinea-pig trachea. Thus, we were interested in investigating whether Bdph at the dose without changing blood pressure antagonized cromakalim-induced systolic pressure reduction in conscious rats. METHODS: Systolic arterial pressures of conscious rats were determined by using the indirect tail-cuff method. RESULTS: Bdph (30 mg/kg, i.p.) did not affect baseline systolic pressure in conscious normotensive and spontaneous hypertensive rats. Bdph (30 mg/kg, i.p.) also did not affect log dose-response curves of prazosin, clonidine and Bay K 8644, a Ca(2+) channel activator, in normotensive rats. However, Bdph (30 mg/kg, i.p.) similar to 4-aminopyridine (4-AP, 0.4 mg/kg, i.p.), a K(+) channel blocker, non-parallelly but surmountably, and partially similar to glibenclamide (GBC, 10 mg/kg, i.v.), an ATP-sensitive K(+) channel blocker, surmountably but not parallelly rightward shifted the log dose-systolic pressure reduction curve of cromakalim, an ATP-sensitive K(+) channel opener, in normotensive rats, respectively. DISCUSSION: The antagonistic effect of Bdph against cromakalim was similar to that of 4-AP, a K+ channel blocker of Kv1 family, and partially similar to that of GBC, an ATP-sensitive K+ channel blocker. Thus, Bdph may be a kind of K+ channel blockers, which have been reviewed to have a potential clinical use for Alzheimer disease. Indeed, Bdph has also been reported to reverse the deficits of inhibitory avoidance performance and improve memory in rats. Recently, 4-AP was reported to treat Episodic ataxia type 2 (EA2) which is a form of hereditary neurological disorder. Consistently, Bdph was recently reported to have antihyperglycemic activity in mice, since GBC is a powerful oral hypoglycemic drug. CONCLUSIONS: Bdph similar to 4-AP and partially similar to GBC may block Kv1 family and ATP-sensitive K(+) channels in conscious normotensive rats.

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 K v 1 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 K v 1 family of K+ channels, to enhance the baseline tension of guinea-pig trachea.

Inhibitory effects of hesperetin derivatives on guinea pig phosphodiesterases and their ratios between high- and low-affinity rolipram binding.

The phosphodiesterase (PDE)4 molecule exists as two distinct conformers, PDE4H and PDE4L , which have high and low affinities, respectively, for the selective PDE4 inhibitor, rolipram. The inhibition of PDE4H and PDE4L is associated with adverse responses, such as nausea, vomiting, and gastric hypersecretion, and with anti-inflammatory and bronchodilator effects, respectively. We determined the therapeutic (PDE4H/PDE4L) ratios of hesperetin-7-O-methylether, hesperetin-5,7,3'-O-trimethylether (HTME), hesperetin-7-O-acetate, hesperetin-7,3'-O-diacetate, hesperetin-5,7,3'-O-triacetate (HTA), hesperetin-5,7,3'-O-tripropionate, hesperetin-5,7,3'-O-tributyrate, hesperetin-5,7,3'-O-triisobutyrate, and hesperetin-5,7,3'-O-tripivatate, and compared these ratios to those of hesperetin, hesperetin-7,3'-O-dimethylether, hesperidin, and hesperidin-3'-O-methylether to identify derivatives with therapeutic ratios and to characterize the structure-activity relationships among these compounds. The activities of PDE isozymes 1 through 5 were measured using a two-step procedure using [(3)H]adenosine 3',5'-cyclic monophosphate or [(3)H]guanosine 3',5'-cyclic monophosphate as substrates. The inhibitory concentration (IC50) for 50% of PDE4 inhibition and effective concentration (EC50) for replacing 50% of [(3)H]rolipram binding on high-affinity rolipram-binding sites was taken as the PDE4L and PDE4H value, respectively. The HTME and the HTA dually inhibited PDE3 and PDE4, and displayed PDE4H/PDE4L ratios of 18.3 and 20.8, respectively, suggesting that they may be candidate drugs for treating asthma and chronic obstructive pulmonary disease (COPD) because the combined inhibition of PDE3 and PDE4 has synergistically anti-inflammatory and bronchodilator effects in COPD patients.

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 IgG(2a) 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 ß-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 (PDE4(H)/PDE4(L)) ratio than hesperidin. Thus, hesperidin-3'-O-methylether may have more potential for use in treating allergic asthma and chronic obstructive pulmonary disease.

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 (PDE4(H)/PDE4(L)) 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.

Hesperetin-7,3'-O-dimethylether selectively inhibits phosphodiesterase 4 and effectively suppresses ovalbumin-induced airway hyperresponsiveness with a high therapeutic ratio.

BACKGROUND: Hesperetin was reported to selectively inhibit phosphodiesterase 4 (PDE4). While hesperetin-7,3'-O-dimethylether (HDME) is a synthetic liposoluble hesperetin. Therefore, we were interested in investigating its selectivity on PDE4 and binding ability on high-affinity rolipram-binding sites (HARBs) in vitro, and its effects on ovalbumin-induced airway hyperresponsiveness in vivo, and clarifying its potential for treating asthma and chronic obstructive pulmonary disease (COPD). METHODS: PDE1~5 activities were measured using a two-step procedure. The binding of HDME on high-affinity rolipram-binding sites was determined by replacing 2 nM [3H]-rolipram. AHR was assessed using the FlexiVent system and barometric plethysmography. Inflammatory cells were counted using a hemocytometer. Cytokines were determined using mouse T helper (Th)1/Th2 cytokine CBA kits, and total immunoglobulin (Ig)E or IgG2a levels were done using ELISA method. Xylazine (10 mg/kg)/ketamine (70 mg/kg)-induced anesthesia was performed. RESULTS: HDME revealed selective phosphodiesterase 4 (PDE4) inhibition with a therapeutic (PDE4H/PDE4L) ratio of 35.5 in vitro. In vivo, HDME (3~30 µmol/kg, orally (p.o.)) dose-dependently and significantly attenuated the airway resistance (RL) and increased lung dynamic compliance (Cdyn), and decreased enhanced pause (Penh) values induced by methacholine in sensitized and challenged mice. It also significantly suppressed the increases in the numbers of 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) of these mice. In addition, HDME (3~30 µmol/kg, p.o.) dose-dependently and significantly suppressed total and ovalbumin-specific immunoglobulin (Ig)E levels in the BALF and serum, and enhanced IgG2a level in the serum of these mice. CONCLUSIONS: HDME exerted anti-inflammatory effects, including suppression of AHR, and reduced expressions of inflammatory cells and cytokines in this murine model, which appears to be suitable for studying the effects of drugs on atypical asthma and COPD, and for screening those on typical asthma. However, HDME did not influnce xylazine/ketamine-induced anesthesia. Thus HDME may have the potential for use in treating typical and atypical asthma, and COPD.

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 IgG(2a) level in the serum of these mice. The PDE4(H)/PDE4(L) 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.

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 (IC(50)) 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 IC(50) 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 IgG(2a) level in serum of these mice. The PDE4(H) value of S-petasin was >300 µM; therefore, its PDE4(H)/PDE4(L) 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.

Calcium channel subtypes for cholinergic and nonadrenergic noncholinergic neurotransmission in isolated guinea pig trachea.

The Ca(2+) channel subtypes in the neurotransmission of isolated guinea pig trachea were elucidated by monitoring the effects of specific Ca(2+) channel blockers on cholinergic contractions and nonadrenergic noncholinergic (NANC) relaxation elicited by electrical field stimulation (EFS). In isolated guinea pig trachea, cholinergic contractile responses to low- and high-frequency EFS were inhibited by the selective N-type calcium channel blocker, ω-conotoxin MVIIA. ω-Agatoxin IVA (a selective P-type blocker), ω-conotoxin MVIIC (a nonselective N-, Q-, and P-type blocker), and nifedipine (a selective L-type blocker) were ineffective, whereas Ni(2+) (a T- and R-type blocker) facilitated cholinergic contractions and produced a late contracture when its concentration exceeded 30 µM. The more the concentration of Ni(2+) increased, the greater the number of incidences and the late contracture areas which occurred. Late contracture did not seem to be due to the effects of acetylcholine, tachykinins, or other polypeptides, but disappeared in the absence of indomethacin. The NANC relaxant responses elicited by the low- and high-frequency EFS were inhibited by ω-conotoxin MVIIA or Ni(2+), but unaffected by ω-Agatoxin IVA, ω-conotoxin MVIIC, and nifedipine. In the absence of indomethacin, Ni(2+) did not alter the ω-conotoxin MVIIA (100 nM)-resistant component of cholinergic contraction, but significantly further inhibited that of NANC relaxation. These results suggest that in isolated guinea pig trachea, cholinergic contraction is regulated by N-type calcium channels which may mask T- and R-type calcium channels and may be co-modulated by both, while NANC relaxation is mainly and independently controlled by N-, T-, and R-type calcium channels.

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.

Ayanin, a non-selective phosphodiesterase 1-4 inhibitor, effectively suppresses ovalbumin-induced airway hyperresponsiveness without affecting xylazine/ketamine-induced anesthesia.

In recent in vitro reports, the IC(50) value of ayanin (quercetin-3,7,4'-O-trimethylether) was 2.2microM for inhibiting interleukin (IL)-4 production from purified basophils, and its therapeutic ratio was >19. Therefore, we were interested in investigating the effects on ovalbumin induced airway hyperresponsiveness in vivo, and to clarify its potential for treating asthma. Ayanin (30-100micromol/kg, orally (p.o.)) dose-dependently and significantly attenuated the enhanced pause (P(enh)) value induced by methacholine in sensitized and challenged mice. It also significantly suppressed the increases in total inflammatory cells, macrophages, lymphocytes, neutrophils, and eosinophils, and levels of cytokines, including IL-2, IL-4, IL-5, and tumor necrosis factor (TNF)-alpha in bronchoalveolar lavage fluid of these mice. However, at 100micromol/kg, it significantly enhanced the level of interferon (IFN)-gamma. In addition, ayanin (30-100micromol/kg, p.o.) dose-dependently and significantly suppressed total and OVA-specific immunoglobulin (Ig)E levels in the serum and bronchoalveolar lavage fluid, and enhanced the IgG(2a) level in serum of these mice. In the present results, ayanin did not affect xylazine/ketamine-induced anesthesia, suggesting that ayanin has few or no adverse effects, such as nausea, vomiting, and gastric hypersecretion. In conclusion, the above results suggest that ayanin may have the potential for use in treating allergic asthma.

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.