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.


Introduction
The rhizomes of Ligusticum chuanxiong Hort. (previously named L. wallichii Franch.) and Angelica sinensis Diels. (Apiaceae) have been used by the Chinese for several thousand years. In ancient medical literature, such as Shen-Nung-Pen-Tsao-Ching, the rhizome of L. chuanxiong Hort. was delineated to prevent and restore stroke-induced dyskinesia. We reported that butylidenephthalide (Bdph), a neutral oil constituent of the rhizome, inhibited cyclooxygenase to have antiplatelet effects [1]. Other investigators reported that shimotsu-to, a prescription of traditional Chinese medicine (TCM), had antiproliferative effects in primary cultures of mouse aorta smooth muscle cells [2], mainly due to Cnidium rhizome-derived phthalides, such as senkyunolide, ligustilide, and Bdph [3]. Both antiplatelet and antiproliferative effects of these crude drugs benefit to prevent stroke.
To recover from stroke-induced dyskinesia the damaged nerve cells need to be repaired mainly by themselves. The vasodilating effects of Bdph [4][5][6] improve the circulation and may partially benefit this restoration. Recently, Bdph was reported to provide neuroprotection by reducing the release of various proinflammatory molecules from activated microglia [7]. It is also reported to maintain stem cell pluripotency by activating the Jak2/Stat3 pathway and increasing the efficiency of induced pluripotent stem cells generation [8]. These results highlight the ability for these crude drugs to aid in the recovery from dyskinesia. Interestingly, Bdph was also reported to inhibit growth of malignant brain tumor [9], lung adenocarcinoma [10], and glioblastomas [11] with a high therapeutic ratio [12].
Male Hartley guinea-pigs (250∼400 g) were obtained from the Animal Center of the National Science Council (Taipei, Taiwan). The animals were housed in ordinary cages at 22 ± 1 ∘ C with a humidity of 50%∼60% under a constant 12/12-h light/dark cycle and provided with food and water ad libitum. Under a protocol approved by the Animal Care and Use Committee of Taipei Medical University, the following in vitro experiments were performed.

Tracheal Preparation.
The guinea-pigs were sacrificed by cervical dislocation after anesthesia, and their tracheas were removed. Each trachea was cut into six segments. Each segment consisted of three cartilage rings. All segments were cut open opposite the trachealis. The segments were randomized to minimize regional variability. Each segment was tied at one end to a holder via silk sutures, placed in 5 mL of Krebs solution containing indomethacin (3 M) throughout the entire experiment, bubbled with a 95% O 2 and 5% CO 2 mixture at 37 ∘ C, and attached by the other end to a force displacement transducer (Grass FT03) for the isometric recording of tension changes on a polygraph (Gould RS3200). The composition of the Krebs solution was (mM): NaCl 120, KCl 4.7, MgSO 4 0.5, KH 2 PO 4 1.2, CaCl 2 2.5, NaHCO 3 25, and dextrose 11.0. The tissues were suspended under an initial tension of 1.5 g and allowed to equilibrate for 1 h with washing at 15-min intervals. After equilibration, the following experiments were performed.

Bdph Enhanced Baseline Tension but Relaxed Precontraction at High
Concentrations. Bdph (30∼1000 M) or its vehicle (0.03∼1% ethyl alcohol) was cumulatively added to examine the tension change of baseline. After the histamine (10 M)-induced precontraction reached steady state, Bdph (30∼1000 M) or its vehicle was cumulatively added to the organ bath. At the end of experiment, papaverine (0.1 mM) was added to maximally relax the tissue and to standardize the relaxation (100%). The log concentration-response curves of Bdph for both baseline tension and relaxation were constructed.

Bdph Also Antagonized Cromakalim-Induced Relaxation.
After preincubation of Bdph (30∼300 M) or its vehicle for 20 min, histamine (10∼30 M) was added to reach a half-maximal contraction and then cumulatively added cromakalim (0.1∼10 M). At the end of experiment, papaverine (0.1 mM) was added to maximally relax the tissue and to standardize the relaxation (100%). The log concentrationresponse curves of cromakalim in the absence and presence of Bdph were constructed.

Interaction between Bdph and Other K + Channel Blockers to Antagonize Cromakalim-Induced Relaxation.
After preincubation of Bdph (100 or 300 M) and other K + channel blockers, such as GBC (1 M), TEA (8 mM), and 4-AP (5 mM) alone or combination for 20 min, histamine (10∼ 30 M) was added to reach a half-maximal contraction, and then cromakalim (0.1∼10 M) was cumulatively added. At the end of experiment, papaverine (0.1 mM) was added to maximally relax the tissue and to standardize the relaxation (100%). The log concentration-response curves of cromakalim in the absence and presence of drug(s), such as Bdph and other K + channel blockers, alone or combination, were constructed. Vrp, Nif, diltiazem, nicardipine, or their vehicles were cumulatively added to examine the tension change of baseline in the isolated trachea. Second, after preincubation of Bdph (300 M) or its vehicle for 20 min, histamine (10∼30 M) was added to reach a half-maximal contraction, and then Vrp (0.01∼10 M) or Nif (0.001∼1 M) was cumulatively added. At the end of experiment, papaverine (0.1 mM) was added to maximally relax the tissue and to standardize the relaxation (100%). The log concentration-response curves of Vrp and Nif in the absence and presence of Bdph were constructed. Third, after preincubation of Bdph (300 M), Vrp (1 M), and Nif (0.1 M) alone or combination for 20 min, histamine (10∼30 M) was added to reach a halfmaximal contraction, and then cromakalim (0.1∼10 M) was cumulatively added. At the end of experiment, papaverine (0.1 mM) was added to maximally relax the tissue and to standardize the relaxation (100%). The log concentrationresponse curves of cromakalim in the absence and presence of drugs, such as Bdph, Vrp, and Nif, alone or combination were constructed.

Statistical
Analysis. The tracheal contraction was expressed as percentage of maximal contraction (100%), with some exceptions expressed as tension. However, the tracheal relaxation was expressed as percentage of maximal relaxation induced by papaverine (100%) at the end of experiment. All values are expressed as mean ± SEM, is the number of experiment. Student's unpaired t-test was used for statistical analysis between test and control with values < 0.05 being regarded as significant.

Effects of Bdph on Baseline and Histamine-Induced Precontraction.
The effect of Bdph (30∼1000 M), compared to its vehicle, on the baseline tension in isolated guinea-pig trachea is shown in Figures 2 and 3(b). Bdph (30∼300 M) did not significantly relax the histamine (10 M)-induced precontraction, except at higher concentrations of 600∼1000 M in the tissue (Figure 3(a)). In contrast, Bdph (30∼300 M) significantly enhanced its baseline tension (Figure 3(b)).

Interaction between Bdph and Other Ca 2+ Channel
Blockers to Antagonize Cromakalim-Induced Relaxation. All Ca 2+ channel blockers used did not enhance or reduce the baseline tension of the isolated guinea-pig trachea (data not shown). Bdph did not influence the relaxant effects of Vrp (Figure 9(a)) and Nif (Figure 9(b)) on the histamine-induced precontraction. Vrp (1 M) and Nif (0.1 M) also did not influence the antagonistic effect of Bdph (300 M) against the cromakalim-induced relaxation ( Figure 10).

Discussion
The present results suggest that the enhancement of basal tension by Bdph is unrelated to the release of cholinergic transmitter, leukotrienes, histamine, serotonin, and neuropeptides [18]. It is also unrelated to the release of prostaglandins, as the experiment was conducted throughout in the presence of indomethacin. However, the enhancement was antagonized by cromakalim (3 M), an ATP-sensitive K + channel opener [17], which may increase outflux of K + and hyperpolarize the membrane of tracheal smooth muscle cells and cause relaxation. Furthermore, Bdph (300 M) also antagonized and rightward shifted the log concentration-relaxation curve of cromakalim on histamine-induced precontraction in the isolated guinea-pig trachea ( Figure 6). Thus, Bdph may be a kind of K + channels blockers, which have been reviewed to have a potential clinical use for Alzheimer disease [19]. Indeed, Bdph have been reported to reverse the deficits of inhibitory avoidance performance and improve memory in rats [20]. GBC (1 M), a specific ATP-sensitive K + channel blocker [17], effectively antagonized and rightward shifted the curve of cromakalim. Bdph neither at 100 M nor at 300 M influenced the antagonistic effect of GBC. Also, Bdph at 300 M did not affect the antagonistic effect of TEA (8 mM), a nonselective big (BKca) and intermediate (IKca) conductance Ca 2+ -activated K + channels blocker [21]. However, Bdph at 300 M significantly enhanced the antagonistic effect of 4-AP (5 mM) and rightward shifted the curve in the combination. The antagonistic effect of the combination was almost the sum of individual effects (Figure 8(b)). This result strongly suggests that the mechanism of Bdph may be similar to that of 4-AP to antagonize cromakalim. The mechanism of Bdph was unrelated to Ca 2+ -dependent K + channels, as all Ca 2+ channel blockers did not influence the antagonistic effect of Bdph against cromakalim. Episodic ataxia type 2 (EA2) is a form of hereditary neurological disorder caused by cerebellar malfunction and is characterized by interictal ataxia and frequent attacks of dyskinesia, vertigo, and imbalance [22]. Recently, 4-AP was reported to treat EA2 [23,24]. The targets of 4-AP are K v 1 family of K + channels, possibly the K v 1.5 subtype [25]. Further investigation is needed to determine whether Bdph is useful in treating EA2.
In conclusion, Bdph (30∼300 M) concentrationdependently evoked an enhancement of baseline tension in isolated guinea-pig trachea. The enhancement was antagonized by cromakalim, and Bdph (300 M) also antagonized cromakalim-induced relaxation. Furthermore, Bdph (300 M) and 4-AP (5 mM) in combination rightward shifted the log concentration-response curve of cromakalim and significantly antagonized the cromakalim-induced relaxation. The antagonistic effect of the combination is almost equal to the sum of individual effects. Therefore, 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.

Conflict of Interests
The authors declare that there is no conflict of interests.